Bruce E. Panneton. BEHAVIORAL RESPONSE TO ULTRASOUND
STIMULATION AND TYMPANAL CHARACTERIZATION OF Megacephela
Carolina Carolina L. (Coleóptera: Carabidae). (Under the direction ofDr. Hal J. Daniel)
Department ofBiology, May 2002.
The behavioral response of the tiger beetle, Megacephela Carolina Carolina L. to intense
ultrasound stimulation was studied. These tests utilized a fixed 40 kHz, 150 dB signal to
represent and simulate, generically, the echo locating calls of insectivorous bats, which
are potential predators of the insect. The tests were designed to: 1) determine if the insect
could detect intense ultrasound, 2) investigate the acoustic startle response (ASR)
behavior of the insect and 3) determine the location of the tympanal hearing organs
associated with the ultrasound detection—^through ablation techniques and subsequent
light-microscopic and scanning electron microscopic investigation. Results revealed the
presence of tympanal hearing organs on the first abdominal tergum. The insect does
respond to intense ultrasound stimulation with a host of ASR behaviors, which suggest
the insect has evolved hearing organs as a direct result of the hunting pressures exerted
by insectivorous bats.
BEHAVIORAL RESPONSE TO ULTRASOUND STIMULATION AND TYMPANAL
CHARACTERIZATION OF Megacephela Carolina carotina L. (COLOEPTERA:
CARABIDAE).
A Thesis
Presented to
The Faeulty of the Department ofBiology
East Carolina University
In Partial Fulfillment
Of the Requirements for the Degree
Master of Science in Biology
by
Bruce Edward Panneton
May 2002
BEHAVIORAL RESPONSE TO ULTRASOUND STIMULATION AND TYMPANAL
CHARACTERIZATION OF Megacephela Carolina Carolina L. (COLOEPTERA:
CARABIDAE).
By
Bruce Edward Panneton
THESIS DIRECTOR
COMMITTEE MEMBER
Professor Gerhard W. Kalmus, PhD
COMMITTEE MEMBER
ProfesSbr Marlp^Sprague, PhD
COMMITTEE MEMBER 1 Lqwu<\ • ^OuJiaCSL
Mr. Thomas M. Charles
CHAIR OF THE DEPARTMENT OF BIOLOGY 7
Professor Ronald J. Newton, PhD
DEAN OF THE GRADUATE SCHOOL MMlim
Thomas L.' FélíIbush, PhD
lojsol
DEDICATION
This thesis is dedicated to my little brother, Brad. It is my hope that the mere existence of
this finished product and achievement will light the path to his success in both life and
academics. There is a light at the end of the tunnel Brad and this book is the proofl
ACKNOWLEDGEMENTS
I would first like to thank my loving wife, Laura, for her support during this project. Her
help in the field, in the lab and on the drawing board were instrumental in the completion
of this research. Her dedication to and support of my endeavors is the reason this
document has come to be. With all my love...
I would also like to recognize my parents, Bruce and Melody, for their never-ending
enthusiasm and support ofmy pursuits, in both life and academics.
TABLE OF CONTENTS
CHAPTER I. THE PROBLEM 1
Statement of the problem 1
Importance of the study 2
CHAPTER IL REVIEW OF THE LITERATURE 4
Ultrasound and Echolocation 5
Coleopterans as a Food Source for Insectivorous Bats 7
Tympanal Hearing Organs and Acoustic Startle Response 8
Megacephela Carolina Carolina, L. 11
CHAPTER m. MATERIALS AND METHODS 22
Capture and Keeping ofTiger Beetles 22
Ultrasound Equipment 23
Ultrasound Stimulation Tests 24
Tympanal Ablation 25
Acoustic Startle Behaviors 26
Characterization ofTympana 27
CHAPTER IV. RESULTS AND DISCUSSION 29
Capture and Keeping ofTiger Beetles 29
Ultrasound Generator 31
Hearing Trial Results 32
Characterization of Tympanal Organs 3 5
CHAPTER V. SUMMARY AND CONCLUSIONS 39
Summary of the Study 39
Suggestions for Further Study 40
REFERENCES 54
LIST OF TABLES
TABLE 1. Potential Bat Predators ofMegacephela Carolina Carolina, L. 14
TABLE 2. List of Insects with Tympanal Hearing Organs 19
TABLE 3. Chi Square Analysis 37
LIST OF FIGURES
FIGURE 1. Distribution ofMegacephela Carolina Carolina, L. 16
FIGURE 2. Schemata—Tympanal hearing organ 18
FIGURE 3. Schemata— Chodotonal Sensory Organ 18
FIGURE 4. Schemata— Megacephela Carolina Carolina, L. —adult and larva 21
LIST OF PLATES
PLATE 1. Adult Megacephela Carolina Carolina, L. 43
PLATE 2. Habitat 43
PLATE 3. Habitat substrate 45
PLATE 4. Lab Aquaria 45
PLATE 5. Sonin 45 ® LFltrasonic Tape Measure 47
PLATE 6. Tether attachment 47
PLATE 7. 1 ^ abdominal tergum and pleural process 49
PLATE 8. Right Tympanal Organ 49
PLATE 9. Tethered Flight 51
PLATE 10.1®* abdominal tergum and pleural process 51
PLATE 11. Tympanal surfece 53
PLATE 12. Total Tympanal Ablation 53
CHAPTER I
THE PROBLEM
The purpose of this study was to investigate the behavioral responses of the
tiger beetle Megacephela Carolina Carolina, Linneaus (Coleóptera: Carabidae) to
intense ultrasonic stimulation, similar to the stimulation created by echolocating
bats (Microchiroptera). This study also documents the external region and
morphology of the tympanal hearing organs involved in the detection of
ultrasonic signals.
Statement of the Problem
The sound detecting mechanisms of M Carolina was first documented on
museum specimens of the insect by Spangler in 1988. Ultrasound-timed tympanal
hearing organs have been observed in many different species of insects; mantids,
moths, crickets, lacewings and butterflies, as well as beetles. Although the
location of the hearing organs vary with each insect, the tympanal hearing organ
consists of three fundamental features that are conserved throughout the class: 1)
an area of thinned cuticle, known as the tympanum (the focus of this study), 2) an
air filled sac or tracheal expansion behind the cuticle; and, 3) a chordotonal
sensory organ. The tympanal organs of M Carolina are located on the first
abdominal tergum under the elytra and wings. Tests were performed to determine
if the insects respond to intense ultrasonic stimuli of 40kHz at sound pressure
levels in excess of 100 dB (re 20 pPa).
2
Insects able to detect the ultrasonic emissions of insectivorous bats employ a
host of behavioral responses to the sounds enabling them to increase their chances
of avoiding aerial predation. These defensive, behavioral responses to ultrasonic
stimulation have been collectively labeled as Acoustic Startle Responses (ASR).
Typical ASR behaviors include: body posturing, head rolls toward the sound
source, abdominal contractions and flight pattern changes. These responses can
vary from species to species, yet they seem to remain highly conserved in the
Insecta. The acoustic startle response of M. Carolina to intense ultrasonic
stimulation will be explored and discussed. Identification of acoustic startle
responses will be accepted as an indication that the tiger beetles can detect intense
ultrasoiuc signals.
Importance of the Study
Hearing organs, utilized for predation avoidance, have evolved at least six
separate times in invertebrates. Additionally, there are countless numbers of
insects that employ hearing organs for conspecific communication. This research
studies the behavioral response of a carabid beetle to intense ultrasound
stimulation. The ultrasound stimulation, used in this study, is intended to
represent (generically) the écholocation calls of insectivorous bats. This study
aids in our understanding of predator-prey interactions, invertebrate behavior and
invertebrate bioacoustics and communication.
3
The results of this research impact many different disciplines, by bringing forth
new questions as well. There are questions regarding the evolution of the
tympanal hearing organs in M. Carolina. There are questions regarding the
potential for conspecific, ultrasonic communication in M Carolina. There are,
also, questions regarding the nocturnal behavior of M. Carolina. Many of these
questions will be posed and addressed later in this work.
CHAPTER II
REVIEW OF THE LITERATURE
The literature reviewed for this investigation begins with ultrasound and bat
écholocation, followed by the food habits of microchiropteran bats. Next, the
literature review will discuss tympanal hearing organs and acoustic startle
response (ASR) behaviors in invertebrate insects, concluding with a description of
the test subject: the carabid beetle, Megacephela Carolina Carolina, L.
Ultrasound sensitive hearing organs have been identified in members of five
insect orders, including the Coleóptera. The other represented orders are:
Lepidoptera, Neuroptera, Orthoptera and Mantodea. Evidence suggests that the
“ears” of these insects have separately evolved in response to the predation
pressures imposed by insectivorous bats that use écholocation while hunting their
prey in the inght skies (Hoy, 1992; Hoy and Robert, 1996). These bats
(Microchiroptera) use ultrasonic sonar to map out their aerial environments and
detect potential prey items, like moths (Roeder and Treat, 1961; Roeder, 1972;
Spangler, 1988b; Connor, 2001), lacewings (Miller, 1970; May, 1991), crickets
(May and Hoy, 1990), mantises (Yager and May, 1990), scarab beetles (Farris,
1994; Forrest et al, 1995; Forrest et al., 1997) and tiger beetles (Spangler, 1988a;
Pennisi, 1996).
5
Ultrasound and Echolocation
Echolocation is a highly specialized form of perception, utilizing neural
translation of high frequency echoes to map nocturnal environments where vision
is limited. The employment of ultrasound, as opposed to audible sound, is very
significant in that ultrasound allows the sender to receive very detailed “glimpses”
of its environment, important when navigating the nocturnal skies. A flying bat
must recognize and avoid large obstacles such as trees and structures and also
recognize small airborne items—its prey. There are about 900 known species of
bats worldwide (Neuweiler, 2000); about 750 of these 900 species are known as
Microchiroptera (small bats). All Microchiroptera use écholocation to perceive
their surroundings and/or himt for prey (Neuweiler, 2000).
Echolocating bats emit high frequency pulse-emissions from their oral and
nasal cavities (Hoy, 1992; Neuweiler, 2000). These emissions (“clicks”) are short
pulses of high frequency sound ranging from 20-200 kHz (Hoy, 1992) at sound
pressure levels that can exceed 100 dB (re 20 pPa) (Griffin, 1984). These intense,
high frequency emissions allow bats to identify objects by size, shape and even
minute details of surface texture (Neuweiler, 2000).
Echolocation is limited, when compared to visual perception under normal
circumstances. Vision, the neural translation of reflected light, gives animals a
continuous image flow of their environments. The translation of ultrasonic
echoes, however, gives pulsed images similar to the visual effects ofa strobe light
(Neuweiler, 2000). Echolocation is also limited to the physical constraints of high
6
frequency sound and the circumstances of its usage. Ultrasonic echoes are usually
focused in the direction of flight and do not translate into broad, panoramic
images. In humans, these acoustic images would be more closely associated with
the visual eifeets ofwearing blinders or tunnel vision.
Echolocation signals range from 80 to 110 dB (re 20 |aPa) with average ranges
usually less than 20 meters and maximum ranges (open-space search signals)
between 50 and 60 meters (Neuweiler, 2000). This range limitation is caused by
two different phenomena. The first is image processing and subsequent
behavioral responses. In order for a bat to receive a single “image” of its
environment it must generate a sound and wait for the corresp)onding echo.
Continuous and timely echo translation is critical to avoiding obstacles and
targeting prey.
The second range limitation is based on the physics of ultrasound, instead of
functional practicality. Ultrasound attenuates rapidly through geometric
attenuation and absorption (Lawrence and Simmons, 1980). Geometric
attenuation means that sound pressure levels decrease with distance from the
soimd source. In free space and ideal conditions without obstacles, sound pressure
levels are decreased by for every doubling of distance from the sound source
(Ewing, 1989). The attenuation of ultrasound is compounded further by other
factors such as sound absorption, which increases with increasing sound
frequency (Lawrence and Simmons, 1980).
7
With all the known limitations to ultrasound and écholocation, it becomes
interesting to ponder how bats evolved these abilities. That is, until you put these
abilities into their naturally occurring context. When compared to vision,
écholocation seems somewhat impotent and useless, until you take away the
benefits of light. By evolving a means ofperception that does not require daylight
to function, echo locating bats were able to take over a niche (the nighttime skies)
dominated by an abundant food source (invertebrates). Additionally, no bats are
blind. If fact, many bats have excellent vision. Visual cues are believed to be used
by bats for orientation and for discerning darkness to determine the appropriate
times to exit and return to their roosts before and after feeding (Whitaker and
Hamilton, 1998).
Coleopterans as a Food Source for Insectivorous Bats
Bats are top-level predators in the night skies. According to McCracken (1986)
20 million Mexican fi-ee-tailed bats Tadarida brasiliensis fi'om one Texas cave
can consume 125 tons of insects in a single night. Eastula and Whitaker (1972)
found that the diets of seven species of insectivorous bats, fovmd in the Big Bend
National Park of Texas, ranged from 6.4 to 44.4% Coleopterans (mostly Carabid
and Scarab beetles). The only other order that consistently outranked the beetles
was Lepidoptera (moths and butterflies).
Coleóptera is the most specious order in the animal kingdom (Wilson and
Peter, 1989; Booth et al., 1990) with over 300,000 known species (Papp, 1984).
8
There are 20 species of bats in the eastern United States (Whitaker and Hamilton,
1998). Nineteen of these 20 species are insectivorous. Of these 19 species of bats,
13 are known or suspected to include beetles in their diets and have habitat ranges
that overlap with the known distribution of M Carolina, the focus of this study
(Knisley and Shultz, 1997; Whitaker and Hamilton, 1998) see Table 1 (potential
predator listing) and Figure 1 (M Carolina distribution).
Vespertilionid, or Mouse-Eared bats account for almost all of these 19
potential coleopteran predators and is the largest family of bats in the world with
318 species worldwide and 14 species in the eastern United States (Whitaker and
Hamilton, 1998). All mouse-eared bats are insectivorous and have highly
developed écholocation abilities. This evidence shows the high level of predation
pressures that can be exerted by the bats on beetles. Thus it stands to reason that
many nocturnal beetles, including M Carolina, would have developed and
evolved ultrasound sensitive hearing structures, similar to other documented cases
in other orders of insects, in order to increase their survivability.
Tympanal Hearing Orçans and Acoustic Startle Response
Insects appear to detect ultrasonic écholocation signals with tympanal hearing
organs (May, 1991; Hoy, 1992; Hoy and Robert, 1996). Tympanal ears are
characterized by three morphological features: 1) an area of thinned cuticle
(tympanal membrane) backed by 2) an air-filled sac or tracheal expansion that
allows the membrane to resonate to sound induced pressure changes, this
9
resonation stimulates 3) an associated chordotonal sensory organ whose scolopoid
bodies terminate in cap cells (Ewing, 1989; Hoy and Robert, 1996). Scolopoid
bodies are individual sensory units within the chordotonal organ. These organs are
associated with the nervous system and work in conjunction with the axons and
dendrites in the delivery of information to the insect brain. Scolopodia can range
from a few to hundreds of units are responsible for the insects’ ability to detect a
wide range of sound frequencies (Fullard and Yack, 1993). Fewer scolopodia
leads to a more narrow range of hearing sensitivity (Ewing, 1989). See Figures 2
and 3 for a generic schematic of a scolopiod body and tympanal hearing organ.
Tympanal organs are pressure receivers. Pressure receivers, by definition,
require a small air space to allow for resonation. Tympanal hearing organs are
believed to have evolved from small tracheal expansions within the insect cuticle
(Blum, 1985). The chordotonal sensory organs associated with these insect “ears”
have been described by Blum (1985) as modified vibrational sensilla. Tympanal
organs we see today could have evolved from proprioceptors (integumental
sensory organs) that may have been adjacent to the ancestral tracheal expansions
mentioned above. These sensilla were most likely stimulated by the sound
pressure-induced resonation of the air space within the expansions, which could
have prompted some of the earliest forms of Acoustic Startle Response (ASR)
behaviors (Blum 1985).
According to Hoy and co-workers (1989), tympanal hearing organs have
evolved, separately, in at least 6 different orders of insects. These tympana have
10
been found in many unique places in different species, yet most of the organs are
tuned to very similar frequency ranges (20-80 kHz), which fall well within the
ultrasonic frequency ranges of foraging bats. Roeder (1972) found that some
sphingid moths have tympana in their mouthparts and respond to acoustic stimuli
between 30 and 70 kHz. Green lacewings {Chrysopa earned) have tympanal
hearing organs in their forewings (Miller, 1970). Some species of crickets have
tympanal organs in their prothoracic legs and respond to ultrasonic acoustic
stimuli between 30 and 90 kHz (Moiseff et al., 1978; May, 1991 ; Hoy and Robert,
1996). Yager and co-workers (1990) found that some mantids have tympana
between their metathoracic legs and respond to ultrasonic stimulations between 20
and 60 kHz. Some scarab beetles have ultrasonic sensitive tympana in the
cervical membrane, that are acoustically tuned to frequencies between 20 and 80
kHz (Forrest et al., 1995; Forrest et al., 1997). Many tiger beetles (Cicindellidae)
have tympana in their first abdominal tergum and respond to acoustic stimuli of
40kHz (Spangler, 1988a) see Table 2 for a listing of insects that utilize tympanal
hearing organs for predator avoidance. Evidence suggests that the ears of these
insects have evolved in response to the predation pressures imposed by
insectivorous bats that use écholocation while hunting their prey in the night skies
(Hoy, 1992).
Different insect orders have evolved diverse yet similar hearing structures to
presumably detect the ultrasonic output of echolocating bats. This hearing ability
has led to the evolution of many stereotypical behavioral responses. This ASR
11
behavior has been documented in Coleóptera, Lepidoptera, Neuroptera,
Orthoptera and Mantodea. Typical ASR behaviors include: body posture changes,
flight pattern changes, abdominal contractions, stop responses, head rolls toward
the soimd source and leg placement changes (Spangler, 1988a; Hoy et al., 1989;
Ewing, 1989; Yager et al., 1990; May, 1991; Hoy, 1992; Farris, 1994; Forrest et
al., 1995 and Forrest et al., 1997).
Megacephela Carolina Carolina, L.
Tiger beetles, the focus of this research, have been found to have tympana
acoustically timed to ultrasonic frequencies (Spangler, 1988a). The research of
Spangler (1988a) and Yager and Spangler (1995, 1997) has shown that many
Cicindella species have tympana broadly tuned to ultrasonic frequencies. The
sensitivity of the organs is greatest around 40 kHz (between 30-60 kHz) wdth
behavioral thresholds averaging 75-80 dB (re 20 pPa). The basic ASR behavior of
the studied tiger beetles, when immobilized, are abdominal contractions (Spangler
1988a). Other, more advanced ASR behaviors were found in Cicindella marutha.
These advanced behaviors include aerodynamic flight changes and even sound
production believed to be designed to “jam” or distort the echo that a foraging bat
would receive, thus aiding in the insects’ escape (Yager and Spangler, 1997).
Although the majority of tiger beetle ASR research has been conducted on the
more common Cicindella species, some mention has been made about the elusive
12
and understudied M Carolina. In his 1988 publication, Spangler noted that
museum specimens ofM. Carolina have enlarged tympana that are:
...close together at the midline of the tergum and
expand both anteriorly and posteriorly, suggesting an
adaptation that would increase sensitivity to lower
frequency sound.
M Carolina is very easily distinguished from members of the Cicindella. Barry
Knisley and Tom Schultz, in The Biology of Tiger Beetles and a Guide to the
species of the South Atlantic States. (1997) describe this insect as:
...body length is 12-20mm. The dorsal surface is
dark metallic green...The ventral surface is metallic green
to dark hluish-black with the posterior abdominal stemite
and lateral portions of the subterminal sclerites yellow-
white to cream colored. Legs, antennae and mouth parts are
light brown... (see Figure 4 for a schematic of M Carolina
and Plate 1 for color photograph ofadult).
M Carolina is nocturnal and gregarious. They seem to prefer very moist
water-edged habitats and can be foimd during the dayhght hours hiding under
debris (see Plates 2 and 3). Unlike many diurnal tiger beetles found in the
southeast, M. Carolina does not usually take flight when disturbed. Instead, they
nm siuprisingly fast. Since these beetles are nocturnal and have the ability to fly,
they are presumed to be targets of insectivorous bats. Thus, the tympana found on
museum specimens by Spangler (1988a) can be presumed to be timed to
ultrasonic frequencies somewhere between 20 and 200kHz and the insects are
presumed to use some form ofASR behavioral response to ultrasonic stimulation.
13
The purpose of the following research is to test these aforementioned
presumptions. Tests will be performed to study the behavioral response of M
Carolina to intense ultrasonic stimulation. Detailed micrographie and
photographic representations of the tympanal region will be included and
discussed.
Table 1. Potential Predators
Bats with habitat ranges that overlap with M Carolina
Scientitic Name Common Name Notes
Myotis austroriparius S.Eastem Myotis Coleopterans = 7.2 - 26.3% of diet
Myotis yyisescens Gray Myotis Scat Studies have revealed Carabid remains
Myotis septentrionalis Northern Myotis Coleopterans are major food source
Myotis sodalis Indiana Myotis Coleopterans are major food source
Pipistrellus subflavus Eastern Pipistrelle Coleopterans are major food source ( 18% Carabids)
Eptesicus fuscm Big Brown Bat Scat Studies: Coleopterans 36.1% (Hamilton 1933)
Lasiurus borealis Eastern Red Bat Coleopterans are major food source
Lasiurus cinéreas Hoary Bat Coleopterans = 20 - 50% of diet depending on age and skill (Rolseth et al. 1994)
Lasiurus intermedius Northern Yellow Bat Coleopterans are major food source
Lasiurus seminolus Seminole Bat Coleopterans = 10% of diet in July and 90% of diet in August
Nycticeius humeralis Evening Bat Scat Studies: Coleopterans 60% (Whitaker and Clem 1992)
Corynorhinus rafmesquii Rafmesque's Big-Eared Bat Lepidopterans are primary food source: 90%
Tadarida brasiliensis Brazilian/Mexican Free-Tailed Bat Coleopterans and Lepidopterans are major food sources
Bats who's habitat ranges have limited overlap with M. Carolina
Scientitic Name Common Name
Myotis leibii Eastern small-footed Myotis
Myotis lucifitgus Little Brown Bat
Lasionycteris noctivagans Silver-Haired Bat
Corynorhinus townsendii Townsend's Big-Eared Bat
Potential Bat predators ofM Carolina Adapted from Whitaker and Hamilton (1998).
15
Figure 1: A map showing the distribution ofhi. Carolina throughout most of
the southern United States—states where M Carolina have been
collected are shaded. Adapted from Knisley and Schultz, 1997.
 
17
Figure 2 and 3: Schematic structure of the tympanal hearing organ (left) found in
the prothoracic leg of a bush cricket and a schematic of a
chordotonal sensesillum—scolopoid body (right). These adapted
schematics are commonly used diagrams found in many works and
adapted, specifically for this literature review, fi-om: Ewing, 1989
(fi-om Schwabe, 1906) and Blum, 1985 (fi-om V.G. Dethier, The
Physiology ofInsects, Methuen, Inc., 1964).
Subgenual Nerve
Subgenual
Organ
Enveloping
Scolopoid
Sense Cell
Table 2. List of Insects with Tympanal Hearing Organs
Order (Familvf Genus snecies Location Ultrasound Soecs References
Ortboptera (Gryllidae) Many jpp. ofcricket Prothoracic Leg 30-90 kHz Moiseffetal. (1978)
(Acrididae) Many spp. ofKatydid Prothoracic Leg * Hoy RR (1992)
Neuroptera Chysopa carnea Fore wing * Miller LA (1975)
Lepidoptera (Sphingidae) Many spp. ofmoths Mouthparts 30-70 kHz RoederKD(1972)
Blattodea Periplanteta americana Ab segments 2-6 * Florentine GJ (1967)
.
Mantodea (Mantidae) Parasphendale agrionina Between Metathor. legs 20-60 kHz +64 dB Yager et al. (1990)
Díptera Ormia ochracea Behind head 5-20kHz Narins(2001)
Coleóptera (Carabidae)
Cicindella marutha 1st ab segment 40 kHz @ 70-75 dB Spangler HG ( 1988), Yager and Spangler ( 1995)
Megacephela Itspp. Museum Specimen Spangle HG (1988)
(Scarabaeidae)
Euetheola spp. Behind the head 20-80 kHz @ <70 dB Forrest TG et al. (1997)
Cyclocephala "spp. ASR 40 kHz "
Dyscinetus 11spp. ASR@40kHz
Oxygrylius "spp. ASR 40 kHz It
This table shows the many different Orders of insects with documented tympanal hearing organs. As you can see,
the 40 kHz signal that used in this study is well within the frequency ranges ofnearly all of the previously studied
animals and should adequately serve the needs of this study.
Specific ultrasound specs were not available
20
Figure 4: Schematic of M. Carolina (B) and side profile of a tiger beetle
larva (A). Art work by Laura A. Otrimski.
 
CHAPTER III
MATERIALS AND METHODS
This study examined the presence of tympanal hearing organs in Megacephela
Carolina Carolina, L. as well as the behavioral response of the insect to intense
ultrasound stimulation. Acoustic startle response behaviors were investigated
using repeated, intense, ultrasound stimuli from a hand-held ultrasound generator.
Ablation tests were performed to determine the location of the suspected
tympanal hearing organs.
Capture and Keeping of Tiger Beetles
All insects were collected between July and September of 1999 and 2000 in
Pitt County, North Carolina and surrounding areas. The specimens were collected
in low-lying moderately wet areas. M. Carolina prefers sandy-soiled environments
with patchy vegetation and open sandy spaces. The beetles can be found during
the daylight hours under bark, wrack, downed tree limbs, tarps and other forms of
litter in the areas mentioned above. The insects were captured by hand and kept
cryogenically in small compartmentalized containers that were transported in
small coolers with frozen ice packs. The extremely low temperatures inside the
coolers induced a temporary state of hibernation and kept the insects from
escaping every time the lid was opened. The insects were kept on ice until they
were taken back to the lab and released into small, plastic aquaria known as
“Kritter Keepers®” (manufactured by Lee’s—size: Kritter Keeper Large
23
Rectangle 14 1/2"L x 9 3/4"H x 8 3/4"W). Some of the insects remained in the
coolers for over two (2) hours. All of the insects (32) were recovered from the
cold transport, in the lab, with no losses.
The aquaria housing the beetles were lined with 1 to 1.25 cm of sand, which
was partially covered with moist paper towels. The paper towels and small
sections of the sand were moistened daily with a spray bottle application of a 1:4
hydrogen peroxide and water solution. This solution helped to limit fungus and
mold growth in the containers. The paper towels served a dual role. First of all,
the paper towel served as cover for the insects to hide under during the day.
Secondly, the paper towels were used as feeding platforms (see Plate 4). The tiger
beetles were fed small portions of lean ground beef every 2-3 days—in the
evenings, just before they became active. The food was placed on the paper
towels and the paper towels were replaced prior to every feeding in order to keep
any mold and fungal growth (on the sand) to a minimum. The aquaria were kept
in cool dry places during the majority of day and periodically placed in
windowsills to keep the containers from becoming too cool.
Ultrasound Equipment
The instrument used to produce the ultrasonic signal was a Sonin 45 ®
ultrasonic tape measure (henceforth referred to as the Sonin45). This hand-held
ultrasoimd generator is designed to give distance measurements up to 45 feet by
projecting a burst of ultrasound and receiving its echo and calculating the time
24
lapsed in between (see Plate 5). The deviee was tested with a Gunner
Rasmussen® model 140BP microphone (flat frequency response 0-100 kHz +/- 3
dB) and analyzed with a Techtronics digital oscilloscope (® Tekscope model
THS 710, 60 MHz). A signal was obtained on the oscilloscope and the period of
the wave and envelope was measured. The measurements revealed that the device
produces a 40 kHz signal with sound pressure levels in excess of 150 dB (re 20
pPa) at 10 cm. This device produces an intense, fixed ultrasonic signal. The
frequency of this signal falls well within the frequency range of many
insectivorous bats (see Table 1) and is very similar to the soimd stimulation levels
used by others (Spangler, 1988; Farris, 1994; Yager and Spangler, 1995; Forrest
et al., 1997).
Ultrasound Stimulation Tests
The insects were glued and immobilized on the end of a stiff copper wire (12-
15cm) with Hotstik® Coolpac© low temperature hot glue. The wire was attached
on one end to the thoracic region of the insects in order to keep their head and
abdominal areas completely mobile and unhindered during the stimulation trials
(see Plate 6). The other end of the wire was connected to a ring stand so that the
beetle could be suspended imder an Olympus ® SZH stereo-zoom microscope.
The microscope and camera equipment was used during the preliminary tests (200
trials) so that the experimenter could ascertain the subtleties ofM carotina’s ASR
behaviors and for documentation. An X-acto® Extra Hands with 2x magnifier.
25
hands free magnifying glass was used, in place of the microscope, for repeated
tests following recognition of the ASR behaviors.
Once the insect was affixed to the wire, the ultrasound tests began. The
Sonin45 was held 10 to 15cm from the tethered insect and turned on by
depressing the power button. Stimulation lasted 2-4 seconds and was repeated 5
times. Each beetle was tested once per day (five trials per test) every 3-5 days.
Testing was performed every 3-5 days, as opposed to every day, in order to try to
keep the insects from becoming accustomed to the Sonin45’s signal.
The beetles were tested in three different ways. The preliminary tests and first
battery of tests were performed on completely intact tiger beetles. The insects
were tethered to the copper wire and tested with no physical alterations (32
subjects: 535 trials). The second battery tests were performed on tethered tiger
beetles with their elytra and wings removed (18 subjects: 90 trials). The final
battery of tests was performed on tiger beetles with complete tympanal ablation
(17 subjects: 85 trials).
Tympanal Ablation
To expose the dorsal, abdominal surface of a beetle the elytra and wings must
be removed. This delicate process begins with holding the insect between the
index finger and thumb. Using forceps, the experimenter grabbed the anal tip of
one elytron and turned the wrist slightly out and gently lifted up on the elytra to
“unlock” the protective covering. The experimenter then pulled the tip forward.
26
laterally, until the elytron was in the fully extended position. Once the elytron was
forced into its fully extended position it will not return to its “closed” position
quickly. At this point, cuticle scissors are used to snip the elytron off at its
connection point on the pleural process. After the elytron was removed, the
cuticle scissors were used to remove the associated wing. This process is repeated
for both elytra and wings. Another method for removing the elytra is by gripping
the tip of one elytron with forceps, as before, and forcing the elytron superiorly,
similar to opening a soda can. This process is much quicker, less subtle and more
destructive to the aforementioned pleural process, which is adjacent to the first
abdominal tergum and is therefore not recommended.
In order to render the tympanal region ineffective a 000 insect pin was inserted
into the tympanal region of the first abdominal tergum and removed, leaving the
tympana ablated. See Plate 12 for an SEM image of an ablated tympanal region.
Acoustic Startle Behaviors
During the acoustic testing, a noticeable behavioral change at the onset of
ultrasound stimulation was assured as a positive indication that M Carolina could
detect intense ultrasound signals. The behaviors of interest were: body posturing,
abdominal contractions, head rolls toward the sound source and stop behaviors
having all been documented as ASR behaviors in other insects. These behaviors
were classified on a graded scale of positive responses, no response (negative)
responses and unknown (questionable) responses.
27
Each tiger beetle was tested 1 to 6 different times depending on the in-lab life
span of the individual and the battery of tests being performed. Chi-Square
analysis was used to analyze the raw data to retain or refute the following null
hypothesis: There is no statistical difference in the behavioral responses of
specimens with intact, suspected, tympanal organs compared to the
behavioral responses of specimens with their suspected tympanal organs
ablated.
Characterization of Tympana
Spangler (1988a) made a note in his work about the location and general shape
of the tympana of M. Carolina. The purpose of these tests was to give precise
measurements and up-close microscopic documentation of the area of thinned
cuticle along the first abdominal tergum of these tiger beetles. Once testing was
completed, or an individual specimen died, the insects were placed in small vials
of Trump’s Fixative, a solution of gluteraldehyde and formaldehyde in a sodium
cacodylate buffer. This fixative kills the insects quickly and preserves them until
they are to be tested. All scanning electron images were taken on a Phillips®
50IB microscope linked to a Gatan Digiscan® and Apple Power Macintosh G4®
computer. The specimens were rinsed twice in 0.2 M sodium cacodylate buffer
for 5 minutes. Excess water was removed by ethanol series dehydration. The
specimens were then dried in a Balzers Union ® CPD 020 critical point drier and
sputter coated with gold palladium alloy by a Teclmics® Hummer V sputter-
28
coater. All other microscopic images were obtained by an Olympus ® C35 AD-2
35 mm camera and exposure control unit attached to an Olympus ® SZH stereo-
zoom microscope.
CHAPTER IV
RESULTS AND DISCUSSION
Capture and Keeping of Insects
In addition to Megacephela Carolina Carolina L., this investigator has
observed various species of diurnal tiger beetles. There are very distinct
differences between Cicindella spp. tiger beetles and M Carolina, especially in
terms of capturing and maintaining the insects. Capturing M Carolina is much
different from capturing diurnal Cicindella spp. To capture diurnal tiger beetles, a
researcher needs a net and quick reflexes along with a little practice. Diurnal tiger
beetles are quick to take flight from perceived threats. They usually do not fly
very far, but they have a tendency to land several feet from where they took off
facing the direction of their perceived threat (personal observation). It is important
for a collector to sneak up on diurnal tiger beetles with the sun in his/her face so a
shadow doesn’t signal the beetle before he/she is close enough take a swipe with
the net. Capturing M Carolina, on the other hand, requires a little persistence,
patience and speed. Since these tiger beetles hide under debris during the day,
sneaking around and solar orientation is not required. As soon as M. Carolina is
uncovered the insect will run, with surprising speed, away from the threat. It is
important to keep up with the insect and make quiek grabs. One should not
attempt to gently grab the insect itself. Instead, it is preferable to grab the insect
and some of the substrate under it. This method serves a dual purpose. First of all,
this method increases the chances of success because instead of trying to grab a
30
moving insect 12-20 mm long, one can grab a piece of palm-sized earth with the
insect in it. Secondly, gabbing soil with the insect decreases the chances of
injuring the tiger beetle.
In addition, maintaining M Carolina, in the lab is much different from diurnal
Cicindella spp. Diurnal tiger beetles prefer warm, weU-lit laboratory settings.
These Cicindella spp. tiger beetles are extremely active during daylight hours. In
a warm, well-lit aquarium it is not uncommon to observe these tiger beetles
running too and from cover flying, fighting, mating or eating. Mold and fungus
growth on these insects is not as much of a threat because they prefer drier
environments. Dehydration, however, is a much greater threat. The water in moist
sand evaporates quickly in a well-lit, warm aquarium. Diurnal tiger beetles can
dehydrate and die in a matter of 1-2 days in the lab if not watered regularly.
Keeping a moist paper towel in the container and soaking one small comer of the
aquarium is sufficient to keep Cicindella spp. tiger beetles hydrated. M Carolina
are completely inactive during the day and will stay under cover for the entire day
if they are not disturbed. As the sun begins to set, the insects begin to move
around in their laboratory aquaria. Because of their preference for lower
temperatures, M. Carolina does not move around quite as fast nor are they as
active as their diurnal cousins. Their preference for moist habitats also promotes
the growth ofmold and fungus, which are both significant threats for these beetles
in the lab. Dark fungus or mold growth is very easy to notice on the legs and
antennae these tiger beetles because of their light colors. By feeding the insects on
31
the paper towels, changing the paper towels regularly and hydrating the soil and
towels (see Plate 4) with a 1:4 peroxide/water solution, mold and fungus growth
can be minimized.
Ultrasound Generator
The Sonin45 is very simple to use and is relatively inexpensive when
compared to other acoustic equipment. It is a very effective tool for producing
high intensity ultrasound. The Sonin45 produces a fixed 40kHz signal with sound
pressure levels in excess of 150 dB (re 20 pPa). Most of the insects studied have
SPL behavioral thresholds of 85 dB (re 20 pPa) or less.
This ultrasound generator will continually send pulsed emissions when holding
down the activation button. The experimenter has used the Sonin45 at night when
bats were flying around a small pond and found that the bats would immediately
turn away fi'om the sound if the generator was activated.
During the preliminary tests, the experimenter noticed that some of the
tethered beetles would show distinctive behavioral responses to the “clicking”
noise made when the activation button on the Sonin45 was pressed. After a few
trials, the activation button was depressed under the table before stimulating M.
Carolina so the tethered insect would not hear the “click.” With the button
depressed the experimenter would bring the apparatus up from under the table and
“sweep” the signal by the insect (on varied sides). This method seemed to be
more representative of the sweeping search patterns used by bats, as they would
32
forage for flying insects. This method produced more pronounced and distinctive
responses and was used throughout all the tests.
Hearing Trial Results
In the hearing trials with elytra intact, 306 out of the 535 trials (57.2%) were
positive responses to ultrasound stimulation. There were 168 negative responses
(31.4%) and 61 unknown or questionable responses (11.4%). The trials with the
elytra removed revealed 72.2% positive responses (65 out of 90 trials). There
were 19 negative responses (21.1%) and 6 unknown or questionable responses
(6.7%). In the total ablation trials, with both tympanal regions destroyed, there
were no positive responses documented out of 85 trials (0.0%). There were,
however, 72 negative responses (84.7%) and 13 unknown or questionable
responses (15.3%) to ultrasound stimulation.
Two hundred preliminary (learning) tests were performed in the late summer
of 1999. Here the experimenter learned behaviors to look for as positive
indications of ultrasound detection. Stop movements and flattening behaviors
were the most easily recognized and common behaviors noticed. Abdominal
contractions, as documented by Spangler (1988a), were very difficult to detect
with these insects.
Stop movements are very easy to recognize. The tethered tiger beetles do one
of three things when they are attached to the copper wire: 1) stand in place
(motionless); 2) run in place; and 3) sometimes fly in place. Stimulation with
33
ultrasound when the insect is running in place, causes the tiger beetle to stop and
remain perfectly still during the first few moments of a continuous signal. When
the insect is flying in place (see Plate 9), ultrasound stimulation causes the beetle
to stop the flight behavior all together. This behavior suggests that écholocation
detection, while in flight, will cause M. Carolina to stop flight and drop to the
groxmd.
The flattening behavior is equally interesting. Sometimes when a tethered,
motionless tiger beetle is stimulated with ultrasound, it will stretch its legs out as
if to flatten its body against the substrate. This behavior suggests that
écholocation detection, while the beetle is standing still, will cause M. Carolina to
flatten its body against the ground in an attempt to diminish its echo against its
surroimdings.
During the subsequent 335 tests, 90 elytra removal tests and 85 ablation tests,
the experimenter began to notice subtle behavioral responses to ultrasoimd
stimulation: antennae movements and abdominal movements. Many times the
experimenter noticed, in association with stop behaviors, the antennae of the
tethered M. Carolina would move in the direction of the sound and follow its
sweeping motion. The abdominal movements were even more pronounced and
more easily noticed when the insect was tethered upside-down. Sometimes the
insects would move the tip of their abdomens in the direction of the sound and
they would also move with the sweeping motion of the soimd stimulus. These
behaviors were noticed when the sound was introduced fi’om either side and fi-om
34
behind. These behaviors suggest the insect can determine sound direction and that
M. Carolina strives to orient its body so as to always be facing away from the
perceived threat. This behavior is similar to the running behavior exhibited by M.
Carolina to visual threats and is a direct contradiction to the avoidance behaviors
exhibited by diurnal Cicindellidae to visual threats in which they fly and land
facing the direction of their enemy (personal observation).
These abdominal and antennae movements could also be visual behavioral
responses. The tiger beetles could very well be visually perceiving the movements
of the Sonin45 (from side introductions) and therefore be trying to run away from
the perceived threat. When the stimulus is introduced from behind the insect,
chances are the beetle cannot see the Sonin45 and is therefore reacting to the
ultrasound itself
Three hundred and seventy one of the 535 trials (57.2%) indicated positive
acoustic startle response behaviors to ultrasound stimulation in intact test subjects.
Seventy two percent of 90 trials indicated positive acoustic startle response
behaviors to ultrasound stimulation in M. Carolina with elytra and wings
removed. Seventy-two of the 85 trials (84.7%) indicated negative acoustic startle
response behaviors to ultrasound stimulation when the suspected tympanal
hearing organs were ablated (the insects used in the elytra removal tests were used
in the ablation tests 1 day later—one insect died overnight). With intact tympana,
M Carolina responded to intense ultrasound stimulation 371 out of 625 trials or
59.3%.
35
Chi-Square analysis revealed a significant difference in the behavioral
responses of specimens with intact tympanal organs compared to the behavioral
responses of specimens with their tympanal organs ablated (X^ = 34.52, df = 2,
p< .001).
This evidence suggests that these tiger beetles do indeed have the ability to
perceive ultrasonic signals and subsequently do employ a host of Acoustic Startle
Responses including: stop behaviors, flattening behaviors, antennae movements
and abdominal movements toward the source. With the tympanal regions
completely ablated, M. Carolina did not show positive ASR behaviors in any of
the 85 trials. This evidence suggests that the area described by Spangler (1988a) is
indeed a tympanal hearing organ.
Characterization of Tympanal Oi^ans
Ablation tests confirm that tympanal hearing organs are present on the first
abdominal tergum of M Carolina (see Plate 12). Plate 7 represents the external
characteristics of the hearing organs. This image shows the larger size of the first
tergum, in relation to the remaining tergii. This size relation can also be seen in
the SEM image of Plate 10. Plate 8 shows the “swollen” nature of the first
abdominal tergum. This “swollen” appearance is a result of the adaptation of
tracheal expansions within the tergum. These tracheal expansions are the air-filled
resonation chambers of the tympanal hearing organ. In Plate 7 distinct tracheal
36
expansions are evident on the first abdominal tergum, separated by the midline of
the insect.
These dark regions are made evident by areas of thinned cuticle (C), the
tympanum, that cover the resonation chambers of the hearing organ. Each
tympanum is very smooth and free of hairs, sensilla, denticles or other sclerites
normally associated with the abdominal tergii of beetles (see Plate 11). This
smoothness and uniformity most likely enables efficient sound detection.
Scanning electron microscopic images and color micrographs were used in
studying both live and dead M. Carolina specimens. Scanning electron
micrographs are instrumental in discerning the surface texture of the tympani.
SEM images give a clear, up-close, monochrome view of the surface of the
tympanal hearing organs of dead specimens. Color micrographs, however, give
indications of texture, thickness, color and subsurface cavities of the live
specimens. These color images are best for displaying the external anatomy of
the tympanal hearing organs for this research.
37
Table 3. Chi-Square Analysis of Data—Long form with Calculations
Responses to Ultrasound Stimulation*
Positive Negative Unknown Total
Intact Tympana 371 (389.96) 187 (164.61) 67 (70.42) 625
Ablated Tympana 72 (53.03) 0 (22.38) 13 (9.57) 85
Total 443 187 80 710
* Data in parentheses, of table, are the calculated Expected Values
Degrees of freedom (df) = (rows - 1) x (columns - 1)
df=(2-l)x(3-l) = 2
Calculating expected frequencies for each cell...
Processing row 1, column 1 ...
Observed value (O) = 371
Expected value (E) = (row total x column total) / grand total
E = (625 X 443) / 710 = 389.964788732394
Chi-square = (O - E) squared / E
Chi-square = ((371 - 389.964788732394) **2) / 389.964788732394
Chi-square = 0.922296633071567
Total chi-square now = 0.922296633071567
Processing row 1, column 2 ...
Observed value (O) =187
Expected value (E) = (row total x column total) / grand total
E = (625 X 187) / 710 = 164.612676056338
Chi-square = (O - E) squared / E
Chi-square = ((187 - 164.612676056338) **2) / 164.612676056338
Chi-square = 3.04467605633803
Total chi-square now = 3.9669726894096
Processing row 1, column 3 ...
Observed value (O) = 67
Expected value (E) = (row total x column total) / grand total
E = (625 X 80) / 710 = 70.4225352112676
Chi-square = (O - E) squared / E
Chi-square = ((67 - 70.4225352112676) **2) / 70.4225352112676
Chi-square = 0.166335211267605
Total chi-square now = 4.1333079006772
Processing row 2, column 1 ...
Observed value (O) = 72
Expected value (E) = (row total x column total) / grand total
E = (85 X 443) / 710 = 53.0352112676056
Chi-square = (O - E) squared / E
Chi-square = ((72 - 53.0352112676056) **2) / 53.0352112676056
Chi-square = 6.78159289023211
Total chi-square now = 10.9149007909093
Processing row 2, column 2 ...
Observed value (O) = 0
Expected value (E) = (row total x column total) / grand total
E = (85 X 187) / 710 = 22.387323943662
Chi-square = (O - E) squared / E
Chi-square = ((0 - 22.387323943662) **2) / 22.387323943662
Chi-square = 22.387323943662
Total chi-square now = 33.3022247345713
Processing row 2, column 3 ...
Observed value (O) = 13
Expected value (E) = (row total x coliann total) / grand total
E = (85 X 80) / 710 = 9.57746478873239
Chi-square = (O - E) squared / E
Chi-square = ((13 - 9.57746478873239) **2) / 9.57746478873239
Chi-square = 1.22305302402651
Total chi-square now = 34.5252777585978
Calculating probability (P)...
Looking up critical values for chi at df= 2:
Sig levels; 0.20 0.10 0.05 0.025 0.01 0.001
Crit vals: 3.22 4.61 5.99 7.38 9.21 13.82
Sig. 0.20: chi is greater than or equal to 3.22
Sig. 0.10: chi is greater than or equal to 4.61
Sig. 0.05: chi is greater than or equal to 5.99
Sig. 0.025: chi is greata- than or equal to 7.38
Sig. 0.01 : chi is greater than or equal to 9.21
Sig. 0.001 : dii is greater than or equal to 13.82
Degrees of freedom: 2
Chi-square = 34.5252777585978
p is less than or equal to 0.001.
The distribution is significant.
Chi-square formatting and calculation proofs were aided by the Web Chi-Square
Calculator software developed and employed by the Georgetown University
Dept ofLinguistics.
www.georgetown.edu/cball/webtools/web_chi.html
CHAPTER V
SUMMARY AND CONCLUSIONS
Summary of the Study
Megacephela Carolina Carolina L. has a pair of tympanal hearing organs on the
first abdominal tergum. Ablation tests confirmed the nature and location of these
organs. Ultrasound stimulation tests have confirmed that these organs are
acoustically tuned to intense ultrasound and the detection of intense ultrasound
elicits Acoustic Startle Response behaviors. Some documented ASR behaviors
include: stop behaviors, flattening behaviors, antennae movements and abdominal
movements toward the ultrasound source. All of this evidence, in addition to the
fact that M Carolina is nocturnal and capable of flight, suggests that M. Carolina
has evolved tympanal hearing organs and ASR behaviors as a defensive
mechanism against insectivorous bat predation.
There is, however, another behavior worthy of noting. M. Carolina is capable
of flying. The insect has functioning wings and elytra that are not fused. Also,
tethered flight behavior has been elicited in the lab. All of this information has
been established by experts and witnessed by the experimenter. Also established
is the fact that M Carolina is not prone to take flight like it’s diurnal cousins.
Perhaps this adaptation is for predator avoidance as well.
It is speculated that this adaptation has come about as a result of the insect’s
habitat selection. Tiger beetles seem to fly with a ballistic trajectory. That is, they
don’t seem to make advanced maneuvers like dipterans or hymenopterans. Taking
40
defensive nocturnal flight with a ballistic trajectory in a water-edged habitat can
lead to life ending landings (in water). Perhaps this behavior is not an adaptation
at all, but a limitation due to flight-thermoregulatory requirements. This behavior
poses a critical question in understanding the acoustic nature ofM Carolina.
Why are M Carolina tiger beetles less likely to take flight as regularly as
diurnal tiger beetles? What is the advantage of being able to detect the ultrasound
of bat sonar when the insect exercises non-flight behaviors? These questions can
be summed up into one question: What is the purpose of M. Carolina’s tympana
and ultrasoimd sensitivity: communication, prey detection, predator avoidance?
Suggestions for Further Research
The following suggestions for further studies are offered by the investigator:
1. The frequency range and behavioral thresholds of M Carolina must be
established (both sonic and ultrasonic). This data will provide critical information
that will enable researchers to better understand the acoustic nature of the insects.
What animals produce sound within those ranges? Are those animals predators or
potential prey? Finding the answers to the above questions will help researchers
understand for what use M. Carolina uses its tympanal hearing organs.
2. Another area of suggested research focuses on the acoustic communication
behaviors of the insects themselves. Does M. Carolina communicate with audible
sound or ultrasound? If so, how does the insect produce the soimd? Is there sexual
dimorphism in the insects’ ability to detect and/or create sound?
41
3. Finally, the flight behaviors and preferences of the insect should be studied.
This research may help in understanding the nature and purpose of the hearing
organs. Is the “flightless behavior” a preference or is it a habitat-specific
adaptation? Is M Carolina subjected to the hunting pressures of bats? If so, is the
flightless behavior and adaptation to avoid bat predation or are the hearing organs
actually used for prey detection or nonspecific communication?
42
Plate 1: Color Photograph of M. Carolina adapted from Knisley and
Schultz (1997). Photograph by T. Koenig, 1994.
Plate 2: Color Photograph ofM. Carolina habitat.
;••K¡r*C«6«OOC',^v|-hl
44
Plate 3: Color photograph ofM Carolina habitat substrate. Note the
overturned debris (tree branch) and litter (plastic bottle). During
the day, M Carolina would be found hiding under these items.
Plate 4: Color photograph ofM Carolina lab aquaria. Note the folded paper
towel that served as a feeding platform, moisture reserve and cover
for the specimens.
 
46
Plate 5: Color Image of the Sonin® 45 Ultrasonic Tape Measure used as
the ultrasound generator.
Plate 6: Micrograph of tethered M. Carolina. All insects were tested under
these conditions. Note the attachment point of the wire tether (W)
along the thoracic region. This method of attachment is non-
invasive, removable (low temperature hot glue (G) is easily
removed) and allows for unrestricted movement of the head, legs
and abdomen. 5X
 
48
Plate 7: Micrograph of the first abdominal tergnm and tympanal regions
(Ty) of a live M. Carolina tiger beetle. Note the two dark “spaces”
within the first abdominal tergum. These dark spaces are areas of
thinned cuticle © backed by the air filled resonation chambers of
the tympanal hearing organs. The pleural process is labeled (PP)
and the second abdominal tergum is labeled (2T). lOX
Plate 8: Micrograph of the right tympana (Ty) of a live M. Carolina tiger
beetle. The pleural process is labeled (PP). Again, note the larger
size of the 1^ abdominal tergum in relation to the other 2 tergii in
the picture (2T and 3T). The larger size of the I®* abdominal
segment allows for the air-filled, resonation chamber of each
tympanal organ. Note the area of thinned cuticle (dark spot to the
right of “Ty” label) along the first abdominal segment. This area is
the tympana. 15x.
 
50
Plate 9: Micrograph illustrating the tethered flight behavior ofa live test
subject. The wire tether is labeled (W). The pleural process is
labeled (PP). The first abdominal tergum and tympanal regions are
labeled (Ty). This photograph illustrates the unrestricting nature of
the tether apparatus, which allows complete movement of the head,
legs and abdomen as well as demonstration of the flight behavior.
Flight was achieved by gently blowing on the insect. Stimulation
with ultrasound caused an immediate cease of the flight behavior.
5X
Plate 10: Scanning electron micrograph of the tympanal region (Ty). Note
the large size of the first abdominal tergum in comparison to the
second abdominal tergum (2T) and pleural process (PP). The
dimples in the tympana are the result of the dehydration process
which causes the air-filled cavities of the tympanal organ to
slightly collapse. 200X
 
52
Plate 11: High magnification, Scanning electron micorgraph of the smooth
surface of a tympanum. 880X
Plate 12: Scanning electron micrograph of the ablated tympanal regions of
the 1®* abdominal tergum of the test subject. The pleural process is
labeled (PP) the second and third abdominal tergi are labeled 2T
and 3T, respectively. The lower ablation (Al) was created by
inserting a dissecting pin into the suspected region of the first
abdominal tergum. Because of the size ofthe upper ablation (A2) it
is likely that this ablation occurred as a result of removing the
elytra and wings. 220X
 
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