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The hearing abilities of elasmobranch fishes

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Title:
The hearing abilities of elasmobranch fishes
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English
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Casper, Brandon M
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University of South Florida
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Auditory evoked potential
Shark
Stingray
Underwater sound
Monopole
Dipole
Directional
Dissertations, Academic -- Marine Science -- Doctoral -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: The hearing abilities of elasmobranch fishes were examined in response to several types of stimuli using auditory evoked potentials (AEP). Audiograms were acquired for the nurse shark, Ginglymostoma cirratum, the yellow stingray, Urobatis jamaicensis, in a controlled environment using a monopole underwater speaker. A dipole stimulus was used to measure the hearing thresholds of the horn shark, Heterodontus francisi, and the white-spotted bamboo shark, Chiloscyllium plagiosum. The dipole experiments yielded much lower thresholds than any other experiment, suggesting that this type of sound specifically stimulated the macula neglecta by creating a strong velocity flow above the head of the shark. A shaker table was created to measure the directional hearing thresholds of the C. plagiosum and the brown-banded bamboo shark, C. punctatum. This experiment showed that these sharks could sense accelerations equally in all directions suggesting that they have omnidirectional ears. The results also yielded higher thresholds than with the dipole, suggesting that the macula neglecta was not stimulated as the sharks were being accelerated. An audiogram was also acquired for the Atlantic sharpnose shark, Rhizoprionodon terraenovae, using a monopole speaker in the field. This experiment revealed that the hearing thresholds did not appear to be masked by ambient noise levels, and resulting thresholds yielded the lowest levels detected by any elasmobranch using AEPs. Taken together, these experiments show that sharks are most sensitive to low frequency sounds in the near field and use both their otoconial endorgans as well as the macula neglecta to sense particle motion.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2006.
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Includes bibliographical references.
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by Brandon M. Casper.
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Includes vita.

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oclc - 179648812
usfldc doi - E14-SFE0001768
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The Hearing Abilities Of Elasmobranch Fishes by Brandon M. Casper A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy College of Marine Science University of South Florida Major Professor: David A. Mann, Ph.D. Joseph J. Torres, Ph.D. Phillip J. Motta, Ph.D. Robert E. Hueter, Ph.D. Stephen M. Kajiura, Ph.D. Date of Approval: October 26, 2006 Keywords: auditory evoked potential, shark, stingray, underwater sound, monopole, dipole, directional Copyright 2006 Brandon M. Casper

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ACKNOWLEDGEMENTS This dissertation could not have been completed without the contribution of many individuals. My advisor, Dr. David Mann has been with me every step of the way offering exceptional guidance, support and a calming influence when research didn’t always go “as planned”. We went on several co ol trips and were able to visit the darker side of elasmobranch hearing without losing a valuable professional friendship. Thanks to my committee members: Dr. Jose Torres, Dr. Phil Motta, Dr. B ob Hueter, Dr. Steve Kajiura and the chair person of the committ ee, Dr. Michelle Heupel. They offered excellent ideas and suggestions which ha ve helped to produce a much stronger dissertation. Thanks to the American Elasm obranch Society as well as the College of Marine Science for the awards and fellowshi ps which helped to fund much of this research. The Marine Sensory Ecology La b has been a fun and productive work environment and several members made important contributions to this research. Thanks especially to Mandy Cook, Jim Locascio, Sara h Egner and Randy Hill, though all of the students have had an influence on my work. Thanks to my parents and brother who provided their constant support and also helped to remind me that there is a wondrous world full of baseball, family, gossip and te levision going on outside of this book. Lastly and of course most importantly, thanks to my ever-loving and amazing wife, Erica. She has put up with all of my $%#^ % for the last five years with more support and help than I ever could have asked for. She has also b een there as a calming influence as well as a motivator and this dissertation couldn’t have been completed without her. And, of course thanks to the many sharks, rays and fishes wh ich contributed their time and lives for this research.

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i TABLE OF CONTENTS List of Tables iii List of Figures iv Abstract vi Introduction 1 Chapter 1: The hearing abilities of the nurse shark, Ginglymostoma cirratum and the yellow stingray, Urobatis jamaicensis 14 Abstract 14 Introduction 15 Materials and Methods 17 Results 21 Discussion 22 Chapter 2: Dipole hearing threshol ds in elasmobranch fishes. 42 Abstract 42 Introduction 43 Materials and Methods 45 Results 51 Discussion 52 Chapter 3: Directional hearing abi lities of elasmobranch fishes. 76 Abstract 76 Introduction 77

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ii Materials and Methods 80 Results 87 Discussion 88 Chapter 4: The hearing thresholds of the Atlantic sharpnose shark, Rhizoprionodon terraenovae 107 Abstract 107 Introduction 108 Materials and Methods 111 Results 114 Discussion 114 Conclusion 127 Literature Cited 135 About the Author End Page

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iii LIST OF TABLES Table 1.1 Directional particle motion th resholds for the nurse shark and yellow stingray 28 Table 1.2 X, Y, Z particle accelerations for the nurse shark 30 Table 4.1 Sound pressure to part icle acceleration conversions 119

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iv LIST OF FIGURES I.1 Sound pressure audiograms of elasmobranchs 8 I.2 Particle acceleration a udiograms of elasmobranchs 10 I.3 Inner ear of elasmobranch 12 1.1 Lagoon experimental setup 32 1.2 Auditory evoked potential from a nurse shark 35 1.3 Particle acceleration audi ograms of elasmobranchs 38 1.4 Equivalent sound pressures of the nurse shark audiogram relative to the field attraction experiment sound levels 40 2.1 Dipole experiment setup 58 2.2 Auditory evoked potentials from a bamboo shark 60 2.3 Cranial auditory mapping of the horn shark 64 2.4 Dipole audiograms of the horn shark and white-spotted bamboo Relative to ambient noise levels in the field 67 2.5 All fish dipole audiograms 69 2.6 Particle acceleration audi ograms of elasmobranchs 71 2.7 Bamboo shark audiogram plotted in terms of acceleration, velocity and displacement 73 3.1 Directional shaker table setup 94 3.2 Direction auditory evoked pot entials of the white-spotted bamboo shark 96 3.3 Directional hearing thresholds fo r the white-spotted bamboo shark brown-banded bamboo shark 100

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v 3.4 Directional shaker audiograms fo r the white-spotted bamboo shark brown-banded bamboo shark 105 4.1 Audiogram of the blacktip shark pl otted against field ambient noise levels 121 4.2 Monopole particle acceleration audiograms of elasmobranchs 123 4.3 Equivalent sound pressures of the blacktip shark audiogram relative to the field attraction experiment sound levels 125 C.1 All particle acceleration audiograms for elasmobranchs 133

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vi ABSTRACT The hearing abilities of elasmobranch fishes were examined in response to several types of stimuli using auditory evoked potentials (AEP). Audiograms were acquired for the nurse shark, Ginglymostoma cirratum the yellow stingray, Urobatis jamaicensis in a controlled environment using a monopole underw ater speaker. A dipole stimulus was used to measure the hearing thresholds of the horn shark, Heterodontus francisi and the white-spotted bamboo shark, Chiloscyllium plagiosum The dipole experiments yielded much lower thresholds than any other expe riment, suggesting that this type of sound specifically stimulated the macula neglecta by creating a strong velo city flow above the head of the shark. A shaker table was cr eated to measure the directional hearing thresholds of the C. plagiosum and the brown-banded bamboo shark, C. punctatum This experiment showed that these sharks could sense accelerations equall y in all directions suggesting that they have omnidirectional ears The results also yi elded higher thresholds than with the dipole, suggesting that the macula neglecta was not stimulated as the sharks were being accelerated. An audiogram was also acquired for the Atlantic sharpnose shark, Rhizoprionodon terraenovae using a monopole speaker in the field. This experiment revealed that the hearing thresholds did not appear to be masked by ambient noise levels, and resulting thresholds yi elded the lowest levels detected by any elasmobranch using AEPs. Taken together, th ese experiments show that sharks are most

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vii sensitive to low frequency sounds in the near field and use both their otoconial endorgans as well as the macula neglect a to sense particle motion.

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1 INTRODUCTION Elasmobranch hearing was intensively st udied in the 1960’s and 1970’s due to the interest by the U.S. Navy after World War II. Research was focused on answering three basic questions: 1) Can sharks hear? 2) If s o, what frequencies can they hear and what structures are involved? 3) From what distan ces can sharks detect sounds? This led to a general understanding of the hearing abilities of elasmobranch fishes, just in time for interest to begin to wane in the 1980’s and essentially disappear in the 1990’s until new methods became available to further exam ine existing paradigms and to ask new questions. Prior to the 1960’s, very lit tle was known about hearing in elasmobranchs. Parker (1909) was the first to show a response to sounds in sharks and f ound that by cutting the auditory and lateral lin e nerves sharks would no longer re spond to any acoustic stimuli. In the 1950’s a series of experiments were c onducted which showed that the semicircular canals, along with the lagenar macula and part of the utricular macula were designed for detection of angular accelerations in elasmobranchs while part of the utricular macula, all of the saccular macula and the macula negl ecta responded to vibrations and were the likely inner ear endorgans re sponsible for acoustic det ection (Lowenstein and Sand, 1940; Lowenstein and Roberts, 1950, 1951). Sound is composed of two major com ponents, the propagating sound pressure wave and particle motion. All fishes detect particle motion (the directional component of

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2 sound) with their inner ear otoliths (oto conia in elasmobranchs) which act as accelerometers. Sound pressure, however, can only be detected by fishes which have a pressure-to-displacement trans ducer, usually the swim bladder in some teleost fishes. Some fishes, such as the otophysans have e volved a specialized c onnection between the swim bladder and the inner ear which can transmit the sound pressure signal being detected by the bladder. In the case of the otophysans, modified vertebrae known as the Weberian ossicles have evolved for this function. Elasmobranchs and other fishes without swimbladders or any other kind of hearing specialization can only detect the particle motion component of sound. Audiograms were obtained for several species of sharks (Kritzler and Wood, 1961; Olla, 1962; Banner, 1967; Nelson, 1967; Kelly and Nelson, 1975). Many were calibrated in terms of acoustic pressure, however as sharks do not have a swim bladder or any kind of hearing specializations responsiv e to sound pressure, th ese measurements are only useful in that they provide an estimate of the frequenc y range of sensitivity. These studies included the bull shark, Carcharhinus leucas (Kritzler and Wood, 1961) and a study on the lemon shark, Negaprion brevirostris (Nelson, 1967) (Fig. I.1). The only audiograms which measured acoustic particle motion were with N. brevirostris (Banner, 1967) and horn shark, Heterodontus francisi (Kelly and Nelson, 1975) (Fig. I.2). The anatomy of the shark inner ear has been examined in great detail (Tester et al. 1972; Corwin, 1977) and in many cases has focu sed specifically on the macula neglecta,

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3 as this has been hypothesized to be on e of the primary detectors of sound in elasmobranchs (Fig. I.3). There have been two proposed pathways of sound to the inner ear. The otolithic pathway involves the inner ear otoconia (sacculus, utricle and lagena). Because the density of the shark’s body is approximately equal to the surrounding water, sound essentially travels through the shar k’s body until it comes in contact with structures of a different density in the ear. In teleost fish es these structures are solid calcium carbonate deposits called otoliths a nd in elasmobranchs these structures are called otoconia which are also calcium carbona te, with exogenous siliceous material, but in a gelatinous matrix. As sound travels thr ough the fish body, it comes in contact with these structures which are overlying the sensory hair cells of the inner ear. Since they are denser than the surrounding tissues, they will lag relative to the re st of the body in the sound field. This lag causes a shearing of the hair cells thus stimulating the ear. The non-otolithic pathway involves the macu la neglecta. This is the inner ear endorgan which is found in the posterior can al duct. The macula neglecta is unique among the endorgans as it does not have otoconia associated with it. Instead, the sensory hair cells are overlain by a cupula, very similar in form to a lateral line organ. It has been

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4 hypothesized that sound travels from above th e head, through an ar ea of loose connective tissue above the otic capsule ca lled the parietal fossa, and into the posterior canal duct via the fenestrae ovalis, a membrane which sepa rates the parietal fossa region from the macula neglecta in the duct (Tester et al., 1972; Fay et al., 1974; Corwin, 1977; Corwin, 1981a). As fluid flows across the macu la neglecta, there will be a movement of the cupula causing shearing of the hair cells. The hair cells of the macula neglecta have been examined and are primarily oriented in the dorsal/ventral direction (Corwin, 1978; Corwin, 1981a; Corwin, 1983; Barber et al., 1985 ). This pathway has been tested by either directly vibrating the parietal fossa (Fay et al., 1974) or by directing sounds over the parietal fossa (Corwin 1981a), both while recording directly from the ramus neglectus nerve of the macula neglecta. In both cases stronger responses were obtained from the ramus neglectus with stimulation from the pari etal fossa region compared to stimulation of any other area of th e head of the sharks. Another major topic of elasmobranch hearing research involved acoustic attraction of sharks in the field. Several re searchers found that by using U.S. Navy J-9 or J-11 class speaker and playi ng irregularly pulsed, low fr equency sounds, that sharks could be attracted from distances as great as several hundred meters (Nelson and Gruber, 1963; Richard, 1968; Myrberg et al., 1969; Nelson et al., 1969; Nelson and Johnson, 1972; Myrberg et al., 1972; Myrberg 1978). 18 di fferent species of sharks were attracted by the sounds, though it has been acknowledged that most of the sounds were unnaturally

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5 loud and would probably not exist in the sh arks’ natural environment (Richard, 1968; Myrberg, 1978; Kalmijn, 1988). However, these experiments do support the only laboratory evidence (Nelson, 1967) that shar ks can localize a sound source. Further anatomical studies also show that the three otoconial endorgans have hair cell orientations polarized in many different directions which woul d aid in directional hearing abilities (Barber and Emerson, 1980; Corwin, 1981a). In the 1980’s Corwin and others (Bullo ck and Corwin, 1979; Corwin et al., 1982) were the first to use the auditory brainstem response (ABR) to obtain evoked potentials in several animals including sharks. This is a neurophysiological method for obtaining evoked potential responses from animals in resp onse to acoustic stimuli. This was later modified for use in obtaining audiograms in fishes (Kenyon et al., 1998) and then used to measure the hearing abilitie s of the little skate, Raja erinacea (Casper et al., 2003). The R. erinacea hearing experiments only measured the hearing thresholds with reference to sound pressure, but created a baseline for usi ng ABR, now referred to as auditory evoked potentials (AEP), to measure he aring abilities of elasmobranch s more efficiently than the previous behavioral experiments (Kritz ler and Wood, 1961; Olla, 1962; Banner, 1967; Nelson, 1967; Kelly and Nelson, 1975; Casper et al., 2003). There were four distinct goals for this dissertation. The first goal was to increase the knowledge of hearing thresholds in el asmobranchs by obtaining audiograms in a variety of elasmobranch species. AEPs were used in several locations to measure hearing

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6 in the nurse shark, Ginglymostoma cirratum the yellow stingray, Urobatis jamaicensis the horn shark, Heterodontus francisi the white-spotted bamboo shark, Chiloscyllium plagiosum the brown-banded bamboo shark, Chiloscyllium punctatum and the Atlantic sharpnose shark, Rhizoprionodon terraenovae The second goal was to examine the hear ing responses of elasmobranchs to a dipole stimulus. All previous hearing expe riments, including both acoustic attraction experiments and audiogram experiments, ha ve used underwater speakers which are monopole stimuli. Several researchers have suggested that a dipole stimulus more closely resembles the kind of sounds (i.e. m ovements of a fish through the water) that elasmobranchs could be attracted to when searching for prey (Kalmijn, 1988; Myrberg, 2001; Bass and Clark, 2003). Hearing measurements were obtained using a dipole stimulus for H. francisi and C. plagiosum with AEPs. The third goal was to examine the directional hearing sensitivity in C. plagiosum and C. punctatum Using a shaker table, the sh arks were exposed to whole body accelerations in different directions to dete rmine if they are more sensitive to sounds from a certain direction as would be suggest ed by the dorsally sensitive macula neglecta versus the apparently omnidire ctional otoconia as seen by the hair cell polarities (Barber and Emerson, 1980; Corwin, 1981a). This experi ment is unique in that it creates an artificial type of acceleration to stimulate the sharks’ ears, without the sound pressure component of sound.

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7 The fourth goal was to quantify the type s of sounds elasmobranchs are typically exposed to in the environment. A pressure /velocity probe was used to record sounds from a variety of locations in terms of particle motion. Most recordings in the field are conducted using hydrophones that only measur e sound pressure and are therefore not relevant to classify the types of sounds th at elasmobranchs could detect. Along these same lines, an audiogram was obtained for R. terraenovae in the field to measure the hearing thresholds of this sh ark in the presence of ambient noise levels and in a natural acoustic environment. This shark is also from the same genus as many of the sharks which were observed in the fi eld attraction experiments.

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8 Figure I.1 Elasmobranch audiograms in terms of sound pressure. All audiograms were acquired using classical c onditioning methods except for the little skate, Raja erinacea in which positive reward conditioning (black sq uare) and auditory evoked potentials (AEP) (open square) were used (Casper et al., 2003). The lemon shark, Negaprion brevirostris (black circle) was modified from Nelson (1967), the bull shark, Carcharhinus leucas (black triangle) was modified from Krit zler and Wood (1961) and the horn shark, Heterodontus francisi (black diamond) was modified from Kelly and Nelson (1975).

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9 80 90 100 110 120 130 140 150 160 10100100010000Frequency (Hz)Sound Pressure Level (dB re 1 Pa) H eterodontus f rancisi N egaprion brevirostris Carcharhinus leucas R aja erinacea (AEP) R aja erinacea (Behavioral)

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10 Figure I.2 Particle acceleration audiograms for el asmobranchs. Both audiograms were obtained using classical conditioni ng methods. The lemon shark, Negaprion brevirostris (black circle) was modified from Banner (1967) and the horn shark, Heterodontus francisi (black diamond) was modified from Kelly and Nelson (1975).

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11 1.00E-04 1.00E-03 1.00E-02 1.00E-01 101001000Frequency (Hz)Particle Acceleration (m/s2) Lemon Shark Horn Shark

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12 Figure I.3 Schematic of the inner ear of an elasm obranch. Key features to note include: EP-endolymphatic pore, ED-endol ymphatic duct, PF-parietal fossa, F-fenestrae ovalis, MN-macula neglecta, PCD-posterior canal duct, L-lagena, S-sacculus, U-utricle. The two proposed pathways of sound travel involve 1) direct stimulation of the sensory hair cells of the sacculus, utricle and lagena, and 2) sounds directed from the dorsal surface of the shark traveling through the parietal fossa and fenestrae ovalis and stimulating the sensory hair cells of the macula neglecta locat ed in the posterior canal duct. Modified from Tester et al. (1972).

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13

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14 Chapter 1: Evoked potential audiograms of the nurse shark ( Ginglymostoma cirratum ) and the yellow stingray ( Urobatis jamaicensis ). ABSTRACT The hearing thresholds of the nurse shark, Ginglymostoma cirratum and the yellow stingray, Urobatis jamaicensis were measured using auditory evoked potentials (AEP). Stimuli were calibrated using a pressu re-velocity probe so that the acoustic field could be completely characterized. The resu lts show similar hearing thresholds for both species and similar hearing thresholds to pr eviously measured audiograms for the lemon shark, Negaprion brevirostris and the horn shark, Heterodontis francisi All of these audiograms suggest poor hearing abilities, rais ing questions about fi eld studies showing attraction of sharks to acoustic signals. By extrapolating the particle acceleration thresholds into estimates of their equivalent far-field sound pressure le vels, it appears that these sharks cannot detect most of the sounds th at have been used in previous studies to attract sharks in the field.

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15 INTRODUCTION Audition in elasmobranchs has been wide ly reviewed (Wisby et al., 1964; Popper and Fay, 1977; Corwin, 1981b, 1989; Myrberg, 2 001; Hueter et al., 2004), but few experiments have been conducted during th e last two decades. Early experiments included measurements of the hearing threshol ds of several species (Kritzler and Wood, 1961; Olla, 1962; Banner, 1967; Nelson, 1967; Kelly and Nelson, 1975; Casper et al., 2003), examinations of the anatomy involved in sound detection (Tester et al., 1972; Fay et al., 1974; Corwin, 1977), mapping the audito ry neural pathways (Barry, 1987), and field attraction experiments to determine wh at sounds attract sharks in their natural environments (Nelson and Gruber, 1963; Rich ard, 1968; Myrberg et al., 1969; Nelson et al., 1969; Nelson and Johnson, 1972; Myrberg et al., 1972; Myrberg, 1978). Despite this literature, the overall hearing abilities of this subclass of fishes remain largely unknown. Of the five species of elasmobranchs tested, only two studies on the lemon shark, Negaprion brevirostris (Banner, 1967) and the horn shark, Heterodontus francisi (Kelly and Nelson, 1975) have measured hearing thresh olds with reference to particle motion, while the rest measured the pressure sensit ivity of elasmobranchs (Kritzler and Wood, 1961; Nelson, 1967; Casper et al ., 2003). Sound consists of a propagating sound pressure wave and directional particle motion (for general reviews see Ka lmijn, 1988; Rogers and Cox, 1988; Bass and Clark, 2003; Bass and McKi bben, 2003). In order to detect sound pressure, a pressure-to-displacement transduc er, such as the swim bladder found in many

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16 teleosts, is required. Without an air-filled cavity the otolith organs can theoretically only detect particle motion, which appears to be the case in all elasmobranchs. Particle motion is a directional stimulus that drops off quickly as the distance from the sound source increases. The audiograms of the N. brevirostris and H. francisi show frequency sensitivity from 20 Hz to 1000 Hz with best sensitivities at lower frequencies. In general, their hearing is not very sensitive in comparison to fishes with peripheral hearing adaptations, such as the goldfish (Fay, 1988). Shark hearin g sensitivity is more similar to fishes without swimbladders or other accessory hearin g structures, all of which can only detect particle motion. In the 1960s and 1970s several researcher s used powerful underwater speakers (US Navy J9 and J11) to transmit a wide va riety of sound stimuli into the water in an attempt to determine what kind of sounds at tract sharks in thei r natural environment (Nelson and Gruber, 1963; Ri chard, 1968; Nelson et al., 1969; Myrberg et al., 1969; Nelson and Johnson, 1972; Myrberg et al., 1972). These researchers found that when playing variably pulsed sounds, especially at low frequencies, sharks appeared to be attracted and would orient to these sounds from distances as far as 250m from the speakers. These results appear contradict ory to laboratory expe riments that have suggested poor hearing sensitivity. Additiona lly, shark ear anatomy indicates they should only detect particle motion, which attenuates quickly as the distance from a sound source

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17 increases. These obvious discrepancies indica te that there is still much unknown about the hearing abilities of elasmobranchs and that further research in this sensory modality of elasmobranchs is needed. The goals of this experiment were to measure the hearing sensitivity of the nurse shark, Ginglymostoma cirratum and the yellow stingray, Urobatis jamaicensis to compare their thresholds to those of other el asmobranchs previously tested. These fishes belong to two orders of elasmobranchs, Or ectolobiformes and Myli obatiformes, in which hearing has never been measured. G. cirratum was one of the many species of sharks that appeared when sounds were played in several of the field experiments (Richard, 1968; Myrberg et al., 1969; Nelson et al., 1969). The resulting thresholds obtained in this experiment can be used to predict how far the nurse shark can detect sounds from a source and relate the results to those obtaine d in the field experime nts. Hearing tests were conducted using the auditory evoked potential method (AEP), a neurophysiological method of recording evoked potentials from the brain in response to acoustic stimuli (Kenyon et al., 1998). This method has been us ed to measure hearing thresholds in the little skate, Raja erinacea and results obtained from this technique were similar to those measured with operant conditioning (Casper et al., 2003). MATERIALS AND METHODS

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18 Five each of G. cirratum (0.70m-1.28m precaudal length) and U. jamaicensis (0.15m-0.24m disc width) were caught with large nets while snorkeling in the water (0.5m-3m) surrounding the Keys Marine Lab (Long Key, Florida) during July of 2003. The fishes were held either in holding lagoons (sharks) or in cement tanks (rays) and fed pieces of squid. The cement lagoon used for hearing tests was 37m X 15m with an island (15m X 2m) in the middle (Fig. 1.1A), and had circulating water pumped from the bay just north of the lab. All experiments were conducted in the na rrow canal between the island and the land surrounding the southern portion of the lagoon where the water depth was 1.05m. The sides of the canal were slop ed at an approximate angle of 45 degrees with curved borders leading to a flat bottom of cement (Fig. 1.1B). Experimental procedures followed guidelines for the care and use of animals approved by the Institutional Animal Care a nd Use Committee at University of South Florida (protocol #2118). Each test fish was submerged in wate r containing 0.05 g/L of MS-222 (tricaine methanosulfate) for less than 1 minute and was then placed in stiff plastic mesh holders (2.54cm X 2.54cm holes). These holders were tightened with tie wraps that were tight enough to keep the fish from moving, but did not affect breathing. The restrained fish was then suspended from an aluminum bri dge (stretching over th e lagoon to the island) using elastic cords 0.5m below the water’s su rface. The transdu cer (Aquasonic Tactile Sound Underwater Speaker AQ339, Clark Synthe sis, Littleton, CO USA) was hung with

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19 an elastic cord from a rope tied across the lagoon 1m from the head of the fish. The rope was tied at both ends onto pieces of steel ba r that were sunk into the ground outside the channel to keep any vibrations from th e speaker isolated from the test fish. Wire electrodes (12mm X 28ga low prof ile needle electrode, JARI Electrode Supply, Gilroy, CA USA) were placed subderm ally 1cm posterior to the endolymphatic pores (recording electrode), in the dorsal mu sculature 3 cm anterior to the dorsal fin (reference electrode) and free in the water (ground electrode). The electrodes were connected to a TDT pre-amplif ier (HS4, Tucker Davis Technologies, Gainesville, FL USA) which was then connected by a fibe r-optic cable to a TDT evoked potential workstation (System 2) with TDT BioSig software. Sounds were 50ms pulsed tones shaped with a Hanning window and were presented with a 70ms presentation period (1 4/second). Test fre quencies ranged from 100 Hz-2000 Hz, but AEP signals were only obta ined from fishes up to 1000 Hz. Sounds were attenuated in 6 dB steps beginning at th e loudest level that c ould be generated at each frequency. The AEP waveforms were di gitized at 25 kHz and averaged between 100-1000 times (Fig. 1.2A). More averages are needed as the signal moves closer to the threshold in order to pull the si gnal out of the AEP noise floor. A 2048-point Fast Fourier Transform (FFT ) was used to analyze the AEP signals in the frequency domain. The entire 70 ms window was FFT transformed because in many of the lower frequencies that were test ed the recorded signa l took up the entire

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20 window so this was done at every frequenc y to remain consistent. An AEP was determined to be present if the signal showed a doubling of the sound frequency (e.g. 400 Hz peak when the signal played was 200 Hz) with a peak at least 3 dB above the AEP noise floor. This frequency doubling occurs in all low fre quency fish AEP testing (Mann et al., 2001; Egner and Mann, 2005). The AEP noise floor is estimated from the AEP power spectrum with a window of 100 Hz around the doubling frequency (50 Hz on each side of the peak) (Fig. 1.2B). Following all hearing tests the fish was removed and replaced with a pressure/velocity probe (Uniaxial Pressure/V elocity Probe, Applied Physical Sciences Corporation, Groton, CT USA) that was pos itioned where the head of the fish was previously. The probe c ontained a velocity geophone (sensitivity 9.36 mV/cm/s, bandwidth 100 Hz-1 kHz) and a hydrophone (sensitivity: -186.1 dB re 1 V/Pa, bandwidth 10 Hz-2 kHz), which could simulta neously record sound pressure and particle velocity. Calibration with the geophone was pe rformed in all orientat ions (0 horizontal (X-axis), 90 horizontal (Y-axis), and vertical (Z-axis)) and all calibrations are computed as Root Mean Square (RMS). For clarifica tion, the x-axis is the along-body axis (head to tail), the y-axis is sound left-right axis on th e fish, and the z-axis is the up-down axis. Many researchers have suggested that the inner ears of fishes act as an accelerometer and therefore detect the particle acceleration of sound (Kalm ijn, 1988; Fay and Edds-Walton, 1997; Bass and McKibben, 2003). Therefore, a ll audiograms have hearing thresholds

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21 shown in units of pa rticle acceleration (m/s2). Particle velocities can be converted to accelerations by multiplying the recorded velocity with [2 x frequency]. Background noise was also measured and was consistently below 10-6 m/s2. A two-way repeated measure ANOVA (SigmaStat) was used to co mpare frequency responses between the G. cirratum and U. jamaicensis to determine if the two species had similar hearing thresholds at each frequency. RESULTS AEP audiograms of G. cirratum and U. jamaicensis are plotted along with the audiograms previously obtained from N. brevirostris (Banner, 1967) and H. francisi (Kelly and Nelson, 1975) (Fig. 1.3). Both speci es had their most sensitive hearing at 300 and 600 Hz. The hearing thresholds were not significantly different between G. cirratum and U. jamaicensis at any frequency (p>0.05) (Table 1). The average G. cirratum threshold at 600 Hz was about 1.5 times more sensitive than the stingray. Based on visual inspection, the audiograms of G. cirratum and U. jamaicensis are similar to H. francisi and N. brevirostris at similar frequencies, with the only obvious difference being G. cirratum having greater sensitivity at 600 Hz co mpared to the other elasmobranchs. The audiograms for both the G. cirratum and U. jamaicensis and the sound propagation measurements are plotted using the horizontal component (x-axis) of particle acceleration as measured by the geophone-hydrophone probe. The vertical and 90 directions (yand

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22 z-axes, respectively) yielded smaller particle accelerations compared to the horizontal direction at each frequency (Table 2). DISCUSSION Comparison of elasmobranch audiograms The hearing thresholds for the G. cirratum and U. jamaicensis do not differ significantly from that of H. francisi or N. brevirostris (Banner, 1967; Kelly and Nelson, 1975), suggesting that these species have a simi lar range and sensitivity of hearing. The only obvious difference in hearing is th e very low threshold at 20 Hz in N. brevirostris suggesting that future elasmobranch hearing experiments should include frequencies at least as low as 20 Hz. Corwin (1978) states that active, piscivorous elasmobranchs could have more developed hearing abilities compar ed to benthic species, because of slight modifications in the ear anatomy between eco morphotypes. This is not apparent among these species due to the similar hearing th resholds observed. The overall auditory anatomy of elasmobranchs is similar among species that have been examined, with differences primarily in number s of hair cells, hair cell po larities and size of the macula neglecta epithelium (Corwin, 1978). While it is possible that these vari ations could affect hearing thresholds, it is more likely that they play a larger role in directional hearing abilities (Corwin, 1978). Thus, it seems proba ble that all elasmobranchs should have relatively similar hearing ranges and thresholds.

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23 It has been suggested (Mann et al., 2 001) that audiograms obtained using AEP can underestimate hearing sens itivity compared to behavi oral testing procedures. Therefore, if there are differences between th e two testing methods, it is possible that the actual hearing thresholds of these species could be low enough to detect the field attraction sounds. However, Casper et al. (2003) found similar thresholds in R. erinacea measured with operant methods and AEPs. Kenyon et al. (1998) also found similar thresholds for the goldfish, Carassius auratus when comparing their AEP data to previously existing behavioral thresholds a nd lower AEP thresholds than behavioral in the oscar, Astronotus ocellatus Future experiments in which audiograms obtained using both AEP and classical conditioning for the same shark will be needed to determine if the AEP method does underestimate the hearing abilities. Characterization of the sound field Another consideration involves characte rizing the sound fiel d created in the lagoon. The largest component of sound came from directly in front of the fishes (Table 2), thereby stimulating hair cells which were pol arized in that direc tion. Very little is known about the hair cell polarizations of the inner ear of elasmobranchs. The only data for the sacculus, utricle and lagena are from two skates, Raja ocellata (Barber and Emerson, 1980) and Raja clavata (Lowenstein et al., 1964). Most elasmobranch inner ear research has focused on the macula ne glecta (Tester et al., 1972; Corwin, 1977;

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24 Corwin, 1978; Barber et al., 1985). The s accular macula contains predominantly dorsal/ventral polarized cells with a smalle r portion of the macula oriented in the anterior/posterior direction. The utricular m acula has mostly anterior/posterior polarized cells with some dorsal/ventral. The utricu lar macula and macula neglecta have all dorsal/ventral polarized cells Experimental evidence (L owenstein and Roberts, 1951) has shown that the utricle and lagena are predominantly equilibrium receptors whereas the sacculus and macula neglecta are the mo st likely acoustic/vibrati on detectors. This evidence combined with the known polarizations of the hair cel ls of these end organs in the two skates suggests that most acoustic s timulation in elasmobranchs would occur for sounds above and below the fish (as wa s suggested by Corwin, 1981a), with less stimulation from the front and back, as occurr ed in this current experiment. To resolve the question about whether elasmobranchs resp ond equally to sound from all directions requires testing the response of elasmobranchs to sounds (o r vibration) along different axes. Field attraction experiments These AEP results can also be compared to the field attr action experiments conducted by Myrberg and others (Richar d, 1968; Myrberg et al., 1969; Nelson et al., 1969 ). G. cirratum was attracted in several of the experiments by low frequency, pulsed sounds. Particle accelerations were not m easured, but sound pressure levels were

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25 recorded, which can be used to estimate the accompanying particle acceleration. In a planar propagating wave the sound pressure is proportional to the acoustic impedance multiplied by the particle velocity, p= cv where, p=pressure (Pa) =density of medium (1030 kg/m3) c=speed of sound in the medium (1500 m/s) v= particle velocity (m/s) The particle velocity (again using the values ob tained from the x-axis direction of particle motion) can then be differentiated to calcula te the particle acceleration. Using this relationship we can calculate the equivalent s ound pressures in the far field that would be required to produce particle accelerations meas ured at threshold for the sharks. Although this equation can only work with a plane propagating wave, it provides a useful approximation of sound pressures that would produce equivalent pa rticle accelerations within the hear ing range of G. cirratum at large distances from the source (Fig. 1.4). Based on these equivalent pre ssures, it would app ear that the sound pressures that were played in the field attracti on experiments should not have been loud enough to attract G. cirratum (one of the species observed in many of the attraction experiments) given the AEP data, illustrating a discrepancy between these attraction experiments and the hearing

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26 thresholds measured in this study. Maximum sound levels that were used in the field attraction experiments reached 150 dB re 1Pa (Nelson et al 1969) from 50-200 Hz, which are below the projected SPL thresholds of the G. cirratum It should be noted that this experiment did not test for hearing threshol ds at frequencies as low as those played in the field attraction experiments (frequencie s below 100 Hz) and it is impossible to know from what distances the sharks could even be detecting the sounds (at least 25 m with Myrberg et al. (1969), 20-30m for Nelson et al. (1969) and unknown fo r Richard (1968)). Natural ambient sound levels also rarely re ach the loudest levels played in these attraction experiments. Among the loudest of these natural sounds are fish choruses, which are typically around 140 dB SPL rms from 50-500 Hz (Locascio and Mann, 2005). Therefore, the more likely stimulus for sh ark hearing are fish swimming nearby, which may leave large, low frequency hydrodynamic fi elds (dipole in na ture) that can be detected by the ear and lateral line (Kalmij n, 1988). Actual measurements of particle acceleration in the field to determine how far it propagates are critical for estimating how far a shark could be from a s ound source and still detect it. Future experiments need to address thes e differences including further testing of hearing in species which were attracted to sounds in the field. Audiograms from only four species of elasmobranchs are not sufficien t for quantifying the hear ing abilities of an entire subclass of fishes. Furthermore, ve ry little is known about the propagation of sound particle acceleration in different envi ronments. Equations and models might be

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27 able to predict these physical parameters in open ocean environments, but actual field measurements, especially in shallow water systems, will provide the data needed to compare the results of the attraction studies with those of the laboratory experiments. The technology exists now for measuring part icle motion in the field as well as the laboratory and must be used for all future hearing experiments involving fishes which cannot detect sound pressure.

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28 Table 1.1 Particle velocity thresholds as reco rded from the geophone and the converted particle accelerations (velocity x (2 x frequency)) and corr esponding sound pressures recorded simultaneously with the hydrophone. Thresholds are determined from the xaxis component of the sound field as the y and z axes yielded much smaller particle accelerations. (See Table 2)

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29 Nurse Shark Recorded Particle Velocity (m/s) Converted Particle Acceleration (m/s2) Corresponding Sound Pressure (dB re 1Pa) 100 Hz 7.18x10-5 0.0099 147.15 200 Hz 4.68x10-5 0.0129 139.40 300 Hz 8.97x10-6 0.0037 136.44 400 Hz 2.65x10-5 0.0147 147.83 500 Hz 3.13x10-5 0.0216 137.89 600 Hz 7.80x10-6 0.0065 134.21 800 Hz 1.27x10-5 0.0141 135.24 1000 Hz 3.51x10-5 0.0486 146.29 Yellow Stingray Particle Acceleration (m/s2) Corresponding Sound Pressure (dB re 1Pa) 100 Hz 9.89x10-5 0.0137 153.05 200 Hz 4.39x10-5 0.0124 147.76 300 Hz 2.79x10-5 0.0116 139.45 400 Hz 6.57x10-5 0.0363 151.60 500 Hz 3.20x10-5 0.0221 143.48 600 Hz 1.19x10-5 0.0099 140.23 800 Hz 1.09x10-5 0.0121 141.01 1000 Hz 6.33x10-5 0.0875 151.07

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30 Table 1.2 Directional particle accele rations in each of the thr ee Cartesian directions as well as the magnitude of the three directi ons combined, measured with the geophone for sound presentations at threshold levels for Ginglymostoma cirratum These data show that most of the acoustic energy was along the X-axis, which is equiva lent to the direct path (straight line from the transducer to th e shark’s head). The Y-axis would be sound coming from the left or right of the shark’ s head, and the Z-axis would be sound coming from above the shark’s head. The magnitude is calculated by the following equation: (X2+Y2+Z2).

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31 Frequency (Hz) X-axis acceleration (m/s2) Y-axis acceleration (m/s2) Z-axis acceleration (m/s2) Magnitude of particle acceleration (m/s2) 100 0.0067 0.0001 0.0017 0.0069 200 0.0035 0.0003 0.0007 0.0036 300 0.0008 0.0001 0.0002 0.0008 400 0.0076 0.0002 0.0015 0.0077 500 0.0203 0.0016 0.0042 0.0208 600 0.0060 0.0003 0.0011 0.0061 800 0.0190 0.0065 0.0044 0.0206 1000 0.0346 0.0239 0.0088 0.0430

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32 Fig. 1.1 A. Overhead view of the lagoon setup. B. Cross-sectional view looking directly at the shark. Figures not drawn to scale.

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33 A

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34 B

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35 Fig. 1.2 A. Example of the 400 Hz AEP of Ginglymostoma cirratum in the time domain with particle accel eration at 1.34 m/s2. B. 2048-point Fast Fourier Transform (FFT) of the same AEP from G. cirratum in response to a 400 Hz sound. The arrow indicates the frequency doubling peak which occurs at 800 Hz. A positive detection is when the peak (at twice the frequency played) is at leas t 3 dB above the AEP noise floor. The AEP noise floor is estimated from the AEP pow er spectrum with a window of 100 Hz around the doubling frequency.

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36 -1000 -500 0 500 1000 01020304050 Time (ms)Evoked Potential (nV) A

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37 -260 -240 -220 -200 -180 -160 -140 0100020003000400050006000 Frequency (Hz)Evoked Potential (dBV) B

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38 Fig. 1.3 Particle acceleration audiogram s obtained for the nurse shark, Ginglymostoma cirratum and yellow stingray, Urobatis jamaicensis The thresholds are the particle accelerations recorded from the X-axis. The ac celerations in the Y and Z directions were much smaller than the X leaving the overa ll magnitude of all three directions approximately equal to the X direction. Data from the lemon shark, Negaprion brevirostris (Banner, 1967) and the horn shark, Heterodontus francisi (Kelly and Nelson, 1975) are plotted for comparison. Standard error bars are included for G. cirratum and U. jamaicensis audiograms.

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39 0.0001 0.001 0.01 0.1 101001000Frequency (Hz)Particle Acceleration (m/s2) Nurse Shark Yellow Stingray Lemon Shark Horn Shark

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40 Fig. 1.4 The sound pressure needed to produce pa rticle accelerations equivalent to the nurse shark, Ginglymostoma cirratum audiogram in a plane propagating wave (square symbols). The sound pressure levels used in the field attraction experiments as well as the average sound pressure level of a sciaenid fish spawning chorus (Locascio and Mann, 2005) are plotted for comparison. Distances from the sound source to the hydrophone for measurements of SPL were 1m for Richard ( 1968), Nelson et al. (1969) and this project, while they were made at 18.5m for Myrberg et al. (1969). Sound pr essure audiograms for the nurse sharks are calculated from th e recorded velocities using the equation P= cV (where P=pressure (Pa), =density of the medium (1030 kg/m3), c=speed of sound in medium (1500 m/s), V=velocity (m/s)). Th e pressures were then log transformed to convert to sound pressu re levels (dB re 1 Pa). The sound levels from this experiment as well as the fish spawning choruses and Ne lson et al. (1969) are based on RMS levels. Richard (1968) and Myrberg et al. (1969) sound levels are based on spectrum levels.

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41 100 110 120 130 140 150 160 170 02004006008001000Frequency (Hz)Sound Pressure Level (dB re 1 Pa) Nurse shark audiogram Nelson et al. 1969 Richard 1968 Myrberg et al. 1969 Fish choruses

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42 Chapter 2: Dipole hearing measurements in elasmobranch fishes. ABSTRACT The hearing thresholds of the horn shark, Heterodontus francisi and the white-spotted bamboo shark, Chiloscyllium plagiosum were measured using a uditory evoked potentials (AEP) in response to a dipole sound stimulus. The audiograms were similar between the two species with lower frequencies yielding lo wer particle accelerat ion thresholds. The particle acceleration audiograms showed more sensitive hearing at low frequencies than previous elasmobranch audiograms, except for the lemon shark, Negaprion brevirostris Auditory evoked potential signals were also recorded while the dipole stimulus was moved to different locations above the head and body. The strongest AEP signals were recorded from the area around the parietal fossa, supporting previous experiments that suggested this region is importa nt for elasmobranch hearing. This is the first time that hearing experiments have been conducted us ing a dipole stimulus with elasmobranchs, which more closely mimics the natural sounds of swimming prey.

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43 INTRODUCTION To date, all hearing measurements (K ritzler and Wood, 1961; Olla, 1962; Banner, 1967; Nelson, 1967; Kelly and Nelson, 1975; Casper et al., 2003; Casper and Mann, 2006), as well as field attraction experime nts (Nelson and Gruber, 1963; Richard, 1968; Myrberg et al., 1969; Nelson et al., 1969; Nelson and J ohnson, 1972; Myrberg et al., 1972; Myrberg, 1978) of elasmobranchs ha ve used a monopole sound source (i.e. underwater speaker) as the mode of acousti c stimulus. However, several authors (Kalmijn, 1988; Myrberg, 2001) have suggested that a dipole sound source would be the more appropriate stimulus for measuring elasmobranch hearing as it more closely represents biological sound (Ba ss and Clark, 2003) that these fi shes could be listening for in the natural environment (i.e a swimming fish). A dipole s timulus is dir ectional along the axis of motion and attenu ates as a function of 1/r3 (r = radial distance from sound source) in the near field, wh ile a monopole radiates sound out in all directions equally and attenuates as a function of 1/r2 in the near field. This is important when considering the field attraction experiments in which many species of sharks have been attracted to large underwater speakers (monopoles) pr oducing “stronger-than-natural” stimuli (Kalmijn, 1988). Depending on the frequenc y and intensity, these monopole stimuli could travel potentially hundreds of meters in the far field and s till be detectable by sharks.

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44 Dipole stimuli have been used to measur e responses of the lateral line in bony fishes (Harris and Van Bergeijk, 1962; Denton and Gray, 1983; Karlsen and Sand, 1987; Coombs et al., 1989; Coombs, 1994; Abboud and Coombs, 2000; Kirsch et al., 2002) and elasmobranchs (Bleckmann et al., 1987, 1989; Maruska and Tricas, 2004). The dipole stimulus has not become as commonly used in hearing experiments as the monopole stimulus (e.g.: Coombs, 1994; Coombs a nd Fay, 1997; Braun and Coombs, 2000; Fay et al., 2002) even though it provide s a more biologically rele vant stimulus. The dipole stimulus is usually a small metal or plastic ball attached to a rigid post that is driven by a mechanical shaker. It vibrates along one axis and therefore is highly directional compared to a monopole source. A variation of a dipole stimulus was used to measure the vibration sensitivity of the parietal fossa in sharks (Fay et al., 1974). The parietal fossa is a subdermal area of loose connective tissue dorsal to the inner ear. It has been proposed that this structure could provide a direct pathway for sound tran smission to the macula neglecta endorgan of the inner ear (Tester et al., 1972; Fay et al., 1974; Corwin, 1977; Corwin, 1981a). In these experiments a vibrating rod was used to stimulate the surface of the head while recording microphonic potentials from the ear. Fay et al. (1974) found that vibrations on the parietal fossa produced stronger responses from the ramus neglectus nerve of the macula neglecta than vibrations from other areas around the head.

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45 The following experiments were designed to measure the responses of two shark species, the horn shark, Heterodontus francisi and the white spotted bamboo shark, Chiloscyllium plagiosum to dipole sound stimuli. H. francisi hearing thresholds have been measured with a monopole underwater speaker previously (Kelly and Nelson, 1975), while C. plagiosum is from a family of elasmobranchs (Hemiscylliidae) which have never had their hearing tested. Thes e shark species were chosen due to their demersal life style making them ideal for experiments in which they must remain motionless for long periods of time. Heari ng tests were conducted using the auditory evoked potential method (AEP), a neurophys iological method of recording evoked potentials from the brain in response to acous tic stimuli (Corwin et al., 1982; Kenyon et al., 1998). This method has been used to meas ure hearing thresholds in the littl e skate, Raja erinacea the nurse shark, Ginglymostoma cirratum and the yellow stingray, Urobatis jamaicensis (Casper et al., 2003; Casper a nd Mann, 2006). The first goal was to measure the audiogram of each species usi ng the dipole shaker fixed in one location. The second goal of our experiments was to measure spatial sensitivity of the sound stimulus by moving the dipole to several lo cations above the head and measuring the level of the evoked response. Since the di pole is directional, it allows mapping of responses over a fine spatial scale. MATERIALS AND METHODS

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46 Three H. francisi (63-74 cm total length) and five C. plagiosum (65-78 cm total length) were maintained in aquaria on 12 hour light/dark cycles and were fed squid, Loligo, sp Hearing experiments were conducte d in a sound isolation booth (2.44 m x 2.44 m x 2.23 m) in a large, fiberglass tank (1.96 m x 0.95 m x 0.60 m) with a water depth of 0.5 m (water temp = 21 C, salinity = 32ppt). Th e tank sat on top of a wood pallet separated from the floor of the booth by four vibration isol ation mounts (Tech Products Corporation model #52512). Experime ntal procedures followed guidelines for the care and use of animals approved by the Institutional Animal Care and Use Committee at University of South Florida protocol #2118. Each subject was placed in stiff plastic mesh holders (2.54 cm x 2.54 cm holes). These holders were tightened with tie wrap s that were tight enough to keep the shark from moving, but did not aff ect breathing. The shark was suspended by an elastic cord hooked through the mesh at the head and tail and looped across an aluminum bar held above the tank by two aluminum A-frames. The A-frames were not directly connected to the tank. The sharks were suspended 20 cm be low the surface of th e water (Fig. 2.1). The mechanical shaker (Brel and Kjaer mini -shaker type 4810) was attached to another aluminum bar which was suspended independe ntly from the experimental tank by PVC pipes attached to the walls of the booth. The setup was desi gned so that the shaker could be moved in an x-y plane above the tank. A stainless steel tube (27 cm long, 0.4 cm

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47 diameter) that was threaded at one end and had a PVC ball (1.3 cm diameter) glued to the other end was screwed into the shaker to provide the dipole s timulus (Fig. 2.1). Wire electrodes (12 mm length, 28 gauge low-profile needle electrode, Rochester Electro-Medical, Inc., Tampa, FL USA) were placed subdermally 1 cm posterior to the endolymphatic pores (recording electrode), in th e dorsal musculature 3 cm anterior to the dorsal fin (reference electrode), and free in the water (ground elec trode) (Fig. 2.1). The electrodes were connected to a TDT pre-am plifier (HS4, Tucker Davis Technologies, Gainesville, FL USA) which was then connect ed by a fiber-optic ca ble to a TDT evoked potential workstation (System 2) with TDT BioSig software. Hearing threshold measurements These methods follow those used by Ca sper and Mann (2006) with the exception that they were performed in an audiology boot h rather than outdoors. All sounds were pulsed tones that were 50 ms in duration a nd shaped with a Hanning window (25 ms rise and fall time). Sounds above 20 Hz were de livered with a 70 ms presentation period (14/second), while 20 Hz sounds had a 1000 ms presentation period (1/second). Test frequencies ranged from 20 Hz -2000 Hz (20, 50, 100, 200, 300, 400, 800, 1000, 2000 Hz). Sounds were attenuated in 6 dB steps beginning at the highest level that could be generated at each frequency (Fig. 2.2A). Th e AEP waveforms were digitized at 25 kHz

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48 and averaged between 100-1000 times. More averages are needed as the signal moves closer to the threshold in order to differe ntiate the signal from the AEP noise floor. A 2048-point Fast Fourier Transform (FFT ) was used to analyze the AEP signals in the frequency domain (Fig. 2.2B). The entire 70 ms window was FFT-transformed, because for many of the lower frequencies th at were tested the AEP signal took up the entire window. This was done at every frequenc y for the analysis to remain consistent. An AEP was determined to be present if the recorded signal s howed a doubling of the sound frequency (e.g. a 400 Hz peak when the signal played was 200 Hz) with a peak at least 3 dB above the AEP noise floor. The AEP noise floor is es timated from the AEP power spectrum with a window of 100 Hz around the doubling frequency (i.e. 50 Hz on each side of the peak). This frequency doubling occurs in all low frequency fish AEP testing (Mann et al., 2001; Egner an d Mann, 2005; Casper and Mann, 2006). Following all hearing tests the fish was removed and replaced with a pressure/velocity probe (Uniaxial Pressure/V elocity Probe, Applied Physical Sciences Corporation, Groton, CT USA) that was positi oned where the head of the fish had been. The probe contained a velocity geophone (s ensitivity 212 mV/cm/s, bandwidth 10 Hz-1 kHz) and a hydrophone (sensitivity : -176 dB re 1 V/Pa, bandwidth 10 Hz-2 kHz), which could simultaneously record sound pressure and particle velocity (Figure 2.2C). Calibration with the geophone wa s performed in all orientatio ns (0 horizontal (X-axis), 90 horizontal (Y-axis), and ve rtical (Z-axis)) and all calib rations are computed as the

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49 Root Mean Square (RMS) for the magnit ude of the three axes combined. The hydrophone was omni-directional and therefor e did not need to be measured along different axes. Many researchers have s uggested the inner ear of fishes act as accelerometers and therefore detect acoustic particle acceleration (Kalmijn, 1988; Fay and Edds-Walton, 1997; Braun et al., 2002; Bass and McKibben, 2003). Therefore, all audiograms have hearing thresholds show n in units of par ticle acceleration (m/s2). Particle velocity of tonal signals can be converted to acceleration with the following equation: acceleration=velocity x (2 x x frequency). The acceleration thresholds are also given as a function of the magnitude of the three (X, Y, Z) directions measured. Background noise was also measured and was consistently below 10-7 m/s2. A repeated-measures ANOVA (SigmaStat) was used to compare threshold measurements between H. francisi and C. plagiosum to determine if the two species had similar hearing thresholds at each frequency. Auditory Cranial Mapping The experimental setup for cranial ma pping was exactly the same as with the hearing threshold measurements detailed a bove. Auditory evoked potentials were recorded only at the highest sound levels for 50, 100 and 200 Hz. To determine the area of the head of the shark wh ich produces the strongest AEP, the dipole stimulus which is still suspended above the shark, was moved to specific locations around the shark. These

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50 locations included 1) 5 cm in front of the an terior rostrum of the shark, 2) directly over the anterior rostrum of the shark, 3) 2.5 cm posterior of the rostrum on the dorsal surface, 4) directly over the dorsal surface between the shark’s eyes, 5) directly above the endolymphatic ducts on the dorsal surface, 6) 2.5 cm posterior and dorsal to the endolymphatic ducts, 7) 5 cm posterior and dor sal of the endolymphatic ducts, 8) 2.5 cm lateral to and above the endolymphatic duc ts, 9) 5 cm lateral to and above the endolymphatic ducts, 10) 10 cm lateral to and above the endolymphatic ducts and 11) at the tip dorsal lobe of the caudal fin (Fig. 2.3). As the stimulus was moved over each location the AEP was obtained at the three frequencies. The AEP’s were transformed using a 2048-point FFT to determine their voltage level. Field Measurements of Ambient Noise Particle Acceleration The geophone/hydrophone apparatus was attached to a ring stand which was driven into the sediment in Bayboro Ha rbor, St. Petersburg, Florida, USA (26 44.309N, 082 09.887W) outside of the University of South Florida, College of Marine Science in 1.2 m deep water with a sand bottom approxima tely 5 m from a sea wall (temperature = 30 C, salinity = 36 ppt). This urban location wa s chosen since it is re latively quiet with little boat activity to provide a baseline for a “quiet” environment that sharks have been observed to inhabit. Ten reco rdings of ambient noise were obtained for periods of 10 s

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51 over one hour from 1300-1400 EST at a sample rate of 50 kHz and were analyzed using a 2048-point FFT in MATLAB. RESULTS Auditory evoked potential levels decr eased with decreasing signal level and showed a doubling of the frequency of the test signal (Fig. 2.2). Hearing thresholds were determined for both species of sharks and ar e plotted as audiograms (Fig. 2.4). There was no significant inter-individual difference in the hearing thresholds for both species or between the overall audiograms of the two speci es (p>.05). Both species had their most sensitive hearing at 20 Hz w ith increasing thresholds as the frequency increased. The highest frequencies that could be detected were 200 Hz for C. plagiosum and 300 Hz for H. francisi. The ambient noise measurements meas ured in Bayboro Harbor were also plotted relative to these audiograms. Ambi ent noise levels were greatest at low frequencies and decreased with increasing frequency. Evoked potentials were also recorded as the dipole stimulus was moved across the body of the shark. In both species of shark, the strongest response was obtained when the dipole was located 5 cm posterior to the endolymphatic pores followed by an almost equally strong response at 2.5 cm posterior to the endolymphatic pores (Fig 2.3A). As the stimulus was moved to anterior, post erior and lateral locations the response

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52 diminished (Figs. 2.3B, C). No responses we re obtained when the di pole was located at the rostrum of the shark, lateral to the head or at the caudal fin. DISCUSSION Audiogram analysis The sharks in this study were most sensit ive to the lowest frequencies tested. The only other auditory thresholds obtained from a dipole stimulus in fishes were for the mottled sculpin, Cottus bairdi and goldfish, Carassius auratus (Coombs, 1994). The shark hearing thresholds measured in this study were lower than C. bairdi and C. auratus thresholds at frequencies belo w 200 Hz (Fig. 2.5). Above 200 Hz C. auratus was more sensitive than the two shark species and C. bairdi Dipole hearing data are particularly relevant as it has been sugge sted that a dipole stimulus more closely represents the type of stimulus that fishes with no hearing speci alizations (i.e. swim bl adder/ear connections or auditory bullae), including elasmobranchs, would detect in the environment (Kalmijn, 1988; Myrberg, 2001; Bass and Clark, 2003). Howe ver, it should be noted that there are very few, pure monopole or dipole sound stimuli that exist in nature. Many sounds, including those of struggling fishes, are inhe rently more complex and take the form of multipole stimuli (Kalmijn, 1988). These audiograms were compared with am bient noise levels which were recorded in a shallow (1.2 m), tidally-dominated and urban body of water (Fi g. 2.4). The ambient

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53 noise levels are well below the hearing thresh olds at all frequencies except for 20 Hz for both species of sharks. This low frequency “noise” is likely associated with wind and wave action, which were low at the time of recording (wind speed = <5 knots, wave height = <0.2 m). It has been suggested th at elasmobranchs might orient to biological noise sources in this frequency range, in cluding sounds produced by a wounded fish as well as normal swimming motions of fishes, us ing both the ear and la teral line (Nelson and Gruber, 1963; Banner, 1968, 1972; My rberg, 2001). The ambient noise measurements suggest that it is possible th at some low frequency, biologically produced sounds could be masked by ambient noise levels ( 20 Hz) depending on their distance from the sharks and the intensity of the sounds being produced. This could be even more prevalent during periods of high wind, rain or anthropogenically generated noise such as boat traffic. Biological sounds at higher frequencies, such as calls from soniferous fishes (typically >100 Hz), would apparently not be masked by ambient noise levels under similar conditions, and could be important sound cues for piscivorous elasmobranchs searching for prey. The dipole hearing thresholds of H. francisi and C. plagiosum are most similar to those of N. brevirostris (Banner, 1967) (Fig. 2.6). The thresholds at 20 Hz and the shape of their audiograms are very similar. Howe ver, as the only low frequencies tested for N. brevirostris were 20 Hz and 320 Hz, the shape of the audiogram is only estimated relative to the dipole audiograms based on these two points. The H. francisi dipole audiogram

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54 measured in this study differs from the H. francisi monopole audiogram (Kelly and Nelson, 1975) (Fig. 2.6). The monopole audi ogram shows much higher thresholds relative to the dipole audiogram, which could, in part, be due to the ambient noise levels which were present during these experiments. The ambient noise power spectrum in the H. francisi monopole experiment was similar in leve l to the audiogram at low frequencies (<100 Hz) suggesting that threshol ds may have been masked at those low frequencies. In contrast, the dipole experiments reported here were conducted in a sound dampening chamber with low ambient noise levels, we ll below the dipole thresholds that were obtained. Comparing audiograms collected with audi tory evoked potentials there is a large difference in thresholds of G. cirratum and U. jamaicensis measured with a monopole source (Casper and Mann, 2006) versus the sharks measured with the dipole source (Fig. 2.6). The most likely explanation for the di fferences between these (and possibly Kelly and Nelson’s (1975) H. francisi monopole experiment), even though the same physiological methods were used, is which e ndorgans were being stimulated in each experiment. Previous experiments have show n that the macula neglecta and the sacculus are the primary endorgans for acoustic detect ion in the elasmobranch ear, with some responses obtained from part of the utricle (Lowenstein and Roberts, 1951). The saccular macula has hair cell polarizations in the anterior/posterior as well as dorsal/ventral directions in two species of skates (Lowenstein et al., 1964; Ba rber and Emerson, 1980),

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55 and a 3-dimensional arrangement in N. brevirostris (Corwin, 1981a). The utricular macula has hair cells polarized primarily in the anterior/posterior directions with some hair cell polarizations in the dorsal/ventral directions (Lowen stein et al., 1964; Barber and Emerson, 1980). The macula neglecta is located in the posterior semicirc ular canal. It is connected by the fenestrae ovalis membrane to the parietal fossa, an area of the head composed of loose connective tissue. It has be en suggested that the parietal fossa is the likely pathway for sound travel directly to the macula neglecta endorgan, which has hair cells polarized in the dorsal/v entral direction (Lowenstein an d Roberts, 1951; Tester et al., 1972; Fay et al., 1974; Corwin, 1977, 1978, 1981a; Bullock and Corwin, 1979; Barber et al., 1985). The macula neglecta does not have mass-loading otoconia like the other endorgans that are sensitive to particle acceleration, and is more similar in design to the ampullae of the semi-circular canals or the lateral line organs having a cupulae overlying the hair cells. Th ese organs are stimulated by fluid flowing across them causing a movement of the cupulae relative to the hair cells. Th e lateral line free neuromasts of Xenopus laevis have been shown to be sensi tive to particle velocity and yield a flat particle velocity response from approximately 0.1-80 Hz (Kroese et al., 1978). If the macula neglecta is velocity sensitive, it should show a similar particle velocity threshold response regardless of a change in frequency. When the particle acceleration thresholds of the shark dipole experiment s are converted to particle velocities (acceleration / (2 * frequency)) the data show a flat response with changing

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56 frequencies (Fig. 2.7). Furthermore, when examining the other existing elasmobranch audiograms (Fig. 2.6) there is typically a relati vely flat response in terms of acceleration, supporting acceleration detection by the otocon ia when using a monopole stimulus. It is important to note that this hypothesis assumes that the summed neural response measured by AEPs does not show frequency filtering that may be produced by higher levels of the auditory system. Since the dipole was located closer to the head and/or ear of the sharks compared to the monopole (1m versus <15cm), it is lik ely that the macula neglecta received a stronger effective stimulus from the dipole, since stimulation of the macula neglecta would require relative movement between the parietal fossa and the rest of the chondrocranium. With the monopole located at 1 m from the shark’s head the vertical particle motion would be equivalent over al l parts of the head, and thus would not generate a strong stimulus through the parietal fossa. Auditory cranial mapping Previous work has suggested that the parietal fossa is one of the pathways of sound (Lowenstein and Roberts, 1951; Tester et al., 1972; Fay et al ., 1974; Corwin, 1977; Bullock and Corwin, 1979; Corwin, 1981a). Two experiments found that placing a lead weight over the parietal fossa of a lemon sh ark reduced the acoustic-evoked activity in response to a speaker playing directed sounds over the head (Bullock and Corwin, 1979;

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57 Corwin, 1981a). Fay et al. ( 1974) stimulated the surface of th e head of a shark directly with a vibrating pole and found that the regi on of the parietal fo ssa yielded stronger voltage potentials from the macula neglec ta than any of the surrounding areas of the head. In this study with H. francisi and C. plagiosum the strongest evoked potentials were recorded when the dipole stimulus was located in the re gion above the parietal fossa and just posterior to the pari etal fossa (Fig. 2.3). As the stimulus was moved away from this region the evoked potential voltage d ecreased, adding further evidence that the parietal fossa is a likely pathway fo r sound travel with a local stimulus. This dipole hearing experiment has provide d the first audiograms obtained using a dipole stimulus for any elasmobranch. This is important as a dipole stimulus more closely represents biological sounds which fishes detect. Fu rther evidence has also been provided suggesting that the pari etal fossa region is a likely pathway for sound travel in elasmobranchs. If elasmobranchs orient to dipole stimuli, then they would likely be limited to near-field acoustic detection. This would severely limit the ability of elasmobranchs to track prey base d on far-field acoustic stimuli.

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58 Figure 2.1 Diagram of the dipole hearing setup. Drawing not to scale.

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59 Dipole Stimulus Reference Electrode Tank Shark Suspension Bar Recording Electrode Ground Electrode Amplifier

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60 Figure 2.2 A. Auditory Evoked Potentials (AEPs) from Chiloscyllium plagiosum in response to a 100 Hz signal at four signal le vels. As the signal is decreased in level (particle acceleration m/s2) the AEP signal also decreases unt il it is lost in the noise at 1.0-4 m/s2. B. 2048 Fast Fourier Transfor m (FFT) of the same AEP for C. plagiosum in response to a 100 Hz sound. The arrow indi cates the frequency doubling peak which occurs at 200 Hz. A positive detection is when the peak (at twice the frequency played) is at least 3 dB above the AE P noise floor. The AEP noise floor is estimated from the AEP power spectrum with a window of 100 Hz around the doubling frequency. C. Pressure and particle velocity raw signals as recorded from the pressure/velocity probe. This example of particle velocity has been recorded in the Z-axis. P = pressure. V = velocity.

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61 0 10 20 30 40 50 60 70 Time (ms)18.0-4 9.1-4 4.5-4 1.0-4 1 uVAEP Level (m/s2) A

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62 0 500 1000 1500 2000 0 0.05 0.1 0.15 0.2 Fre q uenc y ( Hz ) Evoked Potential ( V) B

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63 0 10 20 30 40 50 60 70 Time (ms) V P117 dB re 1 Pa 1.6 x 10-5 m/s C

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64 Figure 2.3 A. Overhead view of the horn shark, Heterodontus francisi depicting the different locations which were stimulated by th e dipole stimulus. 1) 5 cm in front of the anterior rostrum of the shark, 2) directly over the anterior rostrum of the shark, 3) 2.5 cm posterior of the rostrum on the dorsal surface, 4) directly over th e dorsal surface between the shark’s eyes, 5) directly above the endol ymphatic ducts on the dorsal surface, 6) 2.5 cm posterior and dorsal to the endolymphatic ducts, 7) 5 cm posterior and dorsal of the endolymphatic ducts, 8) 2.5 cm lateral to a nd above the endolymphatic ducts, 9) 5 cm lateral to and above the endolymphatic duc ts, 10) 10 cm lateral to and above the endolymphatic ducts and 11) at the tip dor sal lobe of the caudal fin. The oval surrounding locations 5, 6 and 7 depicts the areas which yielded the strongest evoked potential from the dipole stimulus. Positions 5 and 6 are the location of the parietal fossa. B and C. Evoked potential levels (mean SD) recorded from H. francisi and the whitespotted bamboo shark, Chiloscyllium plagiosum respectively, at each location for 50, 100 and 200 Hz. Note: the closer the level ob tained in dBV was to 0, the stronger the evoked potential that was recorded. 200 Hz yielded a weaker evoked potential in both species relative to 50 and 100 Hz as it is th e upper range of heari ng in these species. Position numbers correspond to numbers on shark from Figure 2.3A.

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65 23456 7 8 9 A Horn Shark-175 -170 -165 -160 -155 -150 -145 1234567891011 Position on SharkEvoked Potential (dBV) 50 Hz 100 Hz 200 Hz B

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66 Bamboo Shark-175 -170 -165 -160 -155 -150 -145 1234567891011 Position on SharkEvoked Potential (dBV) 50 Hz 100 Hz 200 Hz C

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67 Figure 2.4 Dipole audiograms of the horn shark, Heterodontus francisi (n=3) and the white-spotted bamboo shark, Chiloscyllium plagiosum (n=5). Standard error bars are included. Ambient noise levels found in a quiet, tidal-dominated shallow harbor are plotted for comparison (dashed line). Broadband background noise in the test tank was consistently below 10-7 m/s2.

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68 1.0E-05 1.0E-04 1.0E-03 1.0E-02 1.0E-01 050100150200250300Frequency (Hz)Particle Acceleration (m/s2) Horn Shark Dipole Bamboo Shark Dipole Ambient Noise

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69 Figure 2.5 Comparison of shark dipole partic le acceleration audiograms with dipole audiograms from the goldfish, Carassius auratus (black squares), and the mottled sculpin, Cottus bairdi (black diamonds), which were obtained using classical conditioning (Coombs, 1994).

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70 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00 101001000Frequency (Hz)Particle Acceleration (m/s2) Goldfish (Coombs 1994) Sculpin (Coombs 1994) Horn Shark Dipole Bamboo Shark Dipole

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71 Figure 2.6 Particle acceleration audi ograms of all tested species of elasmobranchs. Nurse shark, Ginglymostoma cirratum and yellow stingray, Urobatis jamaicensis are modified from Casper and Mann (2006), the lemon shark, Negaprion brevirostris from Banner (1967) and the horn shark, Heterodontus francisi (black circle, monopole) from Kelly and Nelson (1975). These four speci es were all tested with a monopole sound stimulus The G. cirratum and U. jamaicensis audiograms were obtained with auditory evoked potentials in terms of particle acceleration. The N. brevirostris and H. francisi audiograms were obtained using classical c onditioning methods with measurements in terms of particle displacement, which was c onverted to particle accelerations in this figure.

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72 1.0E-05 1.0E-04 1.0E-03 1.0E-02 1.0E-01 101001000Frequency (Hz)Particle Acceleration (m/s2) Nurse Shark Yellow Stingray Lemon Shark Horn Shark Horn Shark Dipole Bamboo Shark Dipole

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73 Figure 2.7 Chiloscyllium plagiosum audiogram plotted in term s of A) acceleration, B) velocity and C) displacement. These results su pport that the macula neglecta is a velocity detector as there is a substant ially flat response in terms of particle veloc ity irrespective of the change in frequency (B). For a velocity sensitive organ, if the thresholds are plotted in terms of acceleration (A) there is an increase in threshold with increase in frequency (approximately 6 dB per octave) and a decrease in threshold with increase in frequency when expressed in term s of particle displacement (C).

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74 Acceleration1.00E-05 1.00E-04 1.00E-03 1.00E-02 101001000 Frequency (Hz)Particle Acceleration (m/s2) A Velocity 1.00E-07 1.00E-06 1.00E-05 1.00E-04 101001000 Frequency (Hz)Particle Velocity (m/s) B

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75 Displacement 1.00E-10 1.00E-09 1.00E-08 1.00E-07 101001000 Frequency (Hz)Particle Displacement (m) C

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76 Chapter 3: The directional hearing abilities of two species of bamboo sharks. ABSTRACT Auditory evoked potentials (AEPs) were us ed to measure the directional hearing thresholds of the white-spotted bamboo shark, Chiloscyllium plagiosum and the brownbanded bamboo shark, Chiloscyllium punctatum at four frequencies and seven directions using a shaker table designed to mimic the particle motion component of sound. Over most directions and frequencies there were no significant differen ces in acceleration thresholds, suggesting that the sharks have omni-directional h earing abilities. Goldfish, Carassius auratus were used as a comparison species with specialized hearing adaptations versus sharks with no known adaptations, and were found to have more sensitive directional responses than the sharks Composite audiograms of the sharks were created from the average of a ll of the directions at each frequency and were compared with an audiogram obtained for C. plagiosum using a dipole stimulus. The dipole stimulus audiograms were significantly lower at 50 and 200 Hz compared to the shaker audiograms in terms of partic le acceleration. This differe nce is hypothesized to be a result of the dipole stimulating the macula neglecta, which would not be stimulated by the shaker table.

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77 INTRODUCTION The ability to localize a sound in fishes is very important for the detection of prey and predators, and in some cases for communication. However, the physics of underwater sound present many problems for dir ectional hearing in fi shes. Sound travels approximately five times faster underwater co mpared to in air. The presence of an external ear for catching s ound (Batteau, 1967) as well as having widely separated ears allowing for the detection of time-of-a rrival differences (Thompson, 1882) are adaptations which help land animals orient to a sound source. The ears of fishes, on the other hand, are very close togeth er and have no external meatus. Also, most fishes can only detect lower frequency sounds which ha ve very long wavelengths (Fay 1988). High frequencies have very short wavelengths whic h could potentially be used to determine directionality by the difference in phases detected between the ears, but only a few families of bony fishes can detect sounds at high frequencies (Astrup and Mhl, 1993, 1998; Mann et al., 1996; Mann et al., 1998, Mann et al., 2001). These differences present problems for fishes in trying to localize a s ound source. However, th e sensory hair cells of the inner ear are arranged in distinct patches with the same directional orientation (Flock, 1964; Popper, 1977) and the otoliths ar e also angled in diffe rent planes (Lu and Popper, 1998). There also appears to be a hi gher level of neural directional processing that has been found along the pathways between the auditory nerve a nd the brain of some species of bony fishes (Edds-Walton and Fay, 2002, 2003). These features allow for

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78 directional sensitivity along th e axis of acoustic particle motion, but the extent and importance of this is only known in a few bony fishes and no elasmobranchs. Directional hearing abilities have been m easured in variety of teleost fishes, but has been largely ignored in elasmobranchs. One behavioral experiment (Nelson 1967) showed that the lemon shark Negaprion brevirostris could differentiate between speakers with an error of only 9.50 at a distance of ~2.1 m. Sharks have also been attracted from long distances in response to high levels of erratically pulsed sounds in the field, most likely necessitating directional sensitivity (Nelson and Gruber 1963, Richard 1968, Myrberg et al. 1969, Nelson et al. 1969, Nelson and Johnson, 1972; Myrberg et al. 1972, Myrberg 1978). Several resear chers have suggested that sharks should be able to detect and localize sounds usi ng both their otoconia as well as the non-otolithic macula neglecta (Corwin, 1981a; Corwin, 1989). Due to the dorsal/ventral polarization of the hair cells in the macula neglecta (Cor win, 1978, 1981a, 1983; Barber 1985), it has been hypothesized that elasmobranchs can detect sounds from above the head through the parietal fossa region using the macula neglecta, and from all directi ons using the otoconia in the saccule and utricle. This differential detection could aid sharks in determining the location of a sound stimulus. Casper and Mann (in press) measured the hearing thresholds of two species of sharks using a dipole stimulus (mechanical shak er with a plastic ball attached to a metal rod) rather than the more commonly used monopole underwater speaker as the sound

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79 stimulus. They found that with the dipole stimulus located above the shark’s head, significantly lower thresholds were obta ined compared with monopole experiments (Casper and Mann, 2006). One hypothesis from th is set of experiments was that sharks could better detect sounds from above the head than when the stimulus was anterior to the shark, supporting the idea of the macula neglecta as being a specialized organ for detecting sounds (including hydrodyna mic stimuli) above the shark. A shaker table has been used for measuri ng directional hearing abilities in several species of teleosts (Fay, 1984; Lu et al ., 1996; Fay and Edds-Walton, 1997a, 1997b; Lu et al., 1998; Edds-Walton et al., 1999; Ma a nd Fay, 2002; Edds-Walton and Fay, 2003). This method applies directiona l whole body accelerations to st imulate the inner ears of fishes. As the fish body is being shaken, structures of greater density than the surrounding tissues, such as the i nner ear otoliths (or otoconia in sharks), lag relative to the rest of the fish body. This lag results in a shearing of the attach ed hair cells thereby stimulating the auditory system. The shaker se tup is unique in that it recreates the effects of a sound stimulus with only the particle motion component of the sound and no sound pressure. This goal of these experiments was to determine 1) if sharks are better able to detect sounds from one particular direction and 2) whether a dipol e stimulus produces a stronger auditory response than whole-body acceleration. The directional hearing abilities of two species of shar ks, the white-spotted bamboo shark, Chiloscyllium

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80 plagiosum and the brown-banded bamboo shark, Chiloscyllium punctatum were measured using a shaker table. These two speci es were chosen due to there demersal life style making them ideal for experiments in which they must remain motionless for long periods of time. Particle acceleration thresholds were measured for seven different directions and four different frequencies using auditory evok ed potentials. Finally, hearing measurements were made using a dipole stimulus with C. plagiosum to compare thresholds to those obtained with wholebody acceleration. It was hypothesized that thresholds would be lower with the dipole stim ulus because the macula neglecta, which is not mass-loaded, would not respond to whole body acceleration, but would to the dipole stimulus. MATERIALS AND METHODS Two juvenile C. punctatum (16.2-18 cm total length) and four juvenile C. plagiosum (17-18.4 cm total length) were mainta ined in aquaria on 12 hour light/dark cycles and fed squid. Heari ng experiments were conducted at the University of South Florida, College of Marine Science and follo wed the guidelines for the care and use of animals approved by the Institutional Animal Care and Use Committee at University of South Florida protocol #2118.

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81 Shaker table setup. The directional hear ing experiments were performed on top of a vibration isolation table (Kine tic Systems, Vibraplane 5602) w ith four vibration, isolation mounts (Tech Products Corporation m odel #52512) underneath to minimize low frequency vibrations. A fish was placed in an aluminum dish (20.5 cm diameter, 5 cm deep) and restrained with cable ties that looped through mounting ba ses affixed to the bottom of the dish. The cable ties were tight enough to stop any movements without affecting the breathing of the fish. The dish was held in place by four custom-built electromagnetic shakers surrounding the outside of the dish w ith a fifth, mechanical shaker position below the dish (Brel and Kjaer, mini-shaker t ype 4810). The electromagnetic shakers were constructed from four rod-shaped ma gnets (Amazing Magnets #R2000D, Ni-Cu-Ni plated, 5 cm x 1.2 cm) which were equal di stances apart and were held in place by smaller disk-shaped magnets (1.4 cm diameter x 0.4 cm thick) on the inside of the dish. The external rod magnets were held in the center of spools of coiled wire that were attached to stainless steel plates. The stainless steel plates were in turn attached to the vibration isolation table (Fig. 3.1). Each electromagnetic shaker was connected to an 8 ohm power resistor to keep the coiled wire from overheating. Standard sp eaker wires connected th e resistor and then led back to an amplifier. The four electroma gnetic shakers were used to deliver stimuli in the horizontal (X-Y) plane. In order to driv e the dish in the Z di rection (up and down)

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82 the mechanical shaker was screwed into th e isolation table below the dish. A nylon screw was threaded into the shaker and a sma ll piece of neoprene was glued to the top of the screw. The bottom of the dish rested on the screw. Calibration of the acceleration signals. Two dual axis (X and Y directions) accelerometers (Dimension Engineering, ADXL320 buffered 5g accelerometer, 312 mV/g sensitivity) were glued perpendicular to each other to create one three dimensional accelerometer for calibrating the accelerations in the X, Y and Z directions (Fig. 3.2A). The accelerometer was attached to the bottom of the shaker dish with double-sided tape so that it would be exposed to the same acceler ations as the dish and the fishes. A laser vibrometer (Polytec, CLV1000) was used to calibrate th e accelerometer recordings. Directional hearing threshold experiments. Hearing thresholds were measured using Auditory Evoked Potentials (AEP) and follow similar methods as previous experiments (Casper and Mann 2006; Casper and Mann, in pr ess). Wire electrodes (12 mm length, 28 gauge low-profile needle electrode, Rocheste r Electro-Medical, Inc., Tampa, FL USA) were placed subdermally 1 cm posterior to the endolymphatic pores in sharks (recording electrode), in the dorsal muscul ature 3 cm anterior to dorsal fin (reference electrode), and free in the water (ground elec trode). The electrodes we re connected to a TDT preamplifier (HS4, Tucker Davis Technologies Gainesville, FL USA) which was then

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83 connected by a fiber-optic cable to a TDT e voked potential workstation (System 2) with TDT BioSig software. A MATLAB program was created to produce the accelerations while simultaneously recording the evoked potenti als from the fishes. The program was designed to allow manipulations of both the am plitude and phase of th e signal so that the accelerations were focused in the desired direction. The software displayed the time domain and frequency domain (Fast Fourier Transform) of the acce leration signal as well as the time and frequency domains of the AEP being recorded from the fish in order to monitor that the appropriate frequenc y was being presented and detected. Frequencies tested included 20, 50, 100 and 200 Hz. Higher frequencies above this were tested (300, 400 and 1000 Hz) and yielded no AEPs. All accelerations were pulsed tones that were 400 ms in duration with a 100 ms cosine squared gated window. Signals were delivered at 2.22 presentations pe r second. Accelerations were attenuated in 6 dB steps beginning at the loudest level that could be generated at each frequency. The AEP waveforms were digitized at 25 kHz and averaged between 100-1000 times. More averages are needed as the signal moves closer to the threshold in order to pull the signal out of the AEP noise floor (Fig. 3.2B). Seven different directions were tested for each species of shark. These include 00 (X-axis), 900 (Y-axis), 300, 600, up and down (Z-axis), and the directional vectors between X-and-Z axes and Y-and-Z axes. The X-axis represents the longitudinal axes of

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84 the shark. The Y-axis represents the lateral ax es of the shark. The Z-axis represents the dorsal/ventral axes of the shark. A 2048-point Fast Fourier Transform (FFT) was used to analyze the AEP signals in the frequency domain. An AEP was determin ed to be present if the signal showed a doubling of the sound frequency (e.g. a 400 Hz peak when the signal played was 200 Hz) with a peak at least 3 dB above the AEP noise floor (Fig. 3.2C). The AEP noise floor is estimated from the AEP power spectrum w ith a window of 100 Hz around the doubling frequency (i.e. 50 Hz on each side of the peak). This frequency doubling occurs in all low frequency fish AEP testing (Mann et al. 2001, Egner and Mann 2005, Casper and Mann 2006, Casper and Mann, in press). Dipole hearing measurements. Hearing measurements were also conducted in C. plagiosum with a dipole stimulus. This specie s was only chosen for the dipole hearing experiments because it was hardier than C. punctatum and could survive repeated testing without negative results. The methods and an alysis follow the same methodology as in a previous dipole hearing experi ment (see methods in Casper a nd Mann, in press). In brief, the dipole stimulus consisted of a mechanical shaker (Brel and Kjaer mini-shaker type 4810) with a stainless steel t ube (27 cm long, 0.4 cm diamete r) that was threaded at one end into the shaker and had a PVC ball (1.3 cm diameter) attached to the other end. Dipole hearing experiments were conducte d in a sound isolati on booth (2.44 m x 2.44 m

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85 x 2.23 m) in a large, fiberg lass tank (1.96 m x 0.95 m x 0.60 m) with a water depth of 0.5 m (water temperature = 21 C, salinity = 32 ppt). The ta nk sat on top of a wooden table separated from the floor by four vibration isolation mounts (Tech Products Corporation model #52512). Each shark was wrapped in a fine nylon me sh. These holders were tightened with metal binder clips that were tight enough to keep the shark from moving, but did not affect breathing. The shark was suspended by PVC pipe with a binder clip attached to one end. The PVC pipe was firmly attached to an aluminum bar held above the tank. The sharks were suspended 20 cm below the surface of the water. The electrodes and their placement were identical to the directional hearing e xperiments. The mechanical shaker (Brel and Kjaer mini -shaker type 4810) was attached to another aluminum bar which was suspended independently from th e experimental tank by PVC pipes attached to the walls of the booth. BioSig software (Tucker-Davis Technologies Gainesville, FL USA) was used for the hearing experiments. All sounds were pul sed tones that were 50 ms in duration and shaped with a Hanning window (25 ms rise a nd fall time). Sounds above 20 Hz were delivered with a 70 ms presentation period (14/second), while 20 Hz sounds had a 1000 ms presentation period (1/second). Test fr equencies ranged from 20 Hz-200 Hz (20, 50, 100, 200 Hz). Sounds were attenuated in 6 dB steps beginning at the loudest level that could be generated at each frequency. Th e AEP waveforms were digitized at 25 kHz and

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86 averaged between 100-1000 times. Positive detection of the signals was determined using the same methods as in the direc tional hearing experiments (see above). Following all hearing tests the fish was removed and replaced with a pressure/velocity probe (Uniaxial Pressure/V elocity Probe, Applied Physical Sciences Corporation, Groton, CT USA) that was positi oned where the head of the fish had been. The probe contained a velocity geophone (s ensitivity 212 mV/cm/s, bandwidth 10 Hz-1 kHz) and a hydrophone (sensitivity : -176 dB re 1 V/Pa, bandwidth 10 Hz-2 kHz), which could simultaneously record sound pressure a nd particle velocity. Calibration with the geophone was performed in all orientations (0 horizontal (X-axis) 90 horizontal (Yaxis), and vertical (Z-axis)) and all calibrati ons are computed as the Root Mean Square (RMS) for the magnitude of the three ax es combined. The hydrophone was omnidirectional and therefore di d not need to be measured along different axes. Many researchers have suggested that the inner ea r of fishes acts as an accelerometer and therefore detect acoustic pa rticle acceleration (Kalmij n, 1988; Fay and Edds-Walton, 1997a; Braun et al., 2002; Bass and McKibben, 2003). Therefore, all audiograms have hearing thresholds shown in un its of particle acceleration (m/s2). Particle velocity of tonal signals can be converted to accelerati on with the following equation: acceleration = velocity x (2 x x frequency). The acceleration thresholds ar e also given as a function of the magnitude of the three (X, Y, Z) di rections measured. Ambient noise in the audiology booth was also measured and was consistently below 10-7 m/s2.

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87 Data analysis Particle acceleration thresholds were log transformed to satisfy assumptions of normality. A repeated-measures ANOVA was used to measure differences between species of sharks. Sin ce no differences were detected, the species were pooled and a repeated-measures ANOVA was used to compare the differences between directions among each of the fre quencies and a Tukey post-hoc comparison was used if the ANOVA showed significant di fferences. The repeated-measures ANOVA with a Tukey post-hoc test was also used to test differences between the white-spotted bamboo thresholds obtained with the shaker and those obtained with the dipole stimulus over all frequencies tested. RESULTS Particle acceleration thre sholds were obtained from both species of bamboo sharks over all seven directions (Fig. 3.3). There was no significant difference between species of sharks (p=0.42), th erefore species were pooled toge ther for testing differences between frequencies and directions. Th ere was no significant difference between directions for each of the individual sharks (p=0.06). There was a significant interaction among direction and frequency, but a Tukey post-hoc test revealed no significant difference among hearing thresholds at any of th e directions tested for any of the species (p>0.05).

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88 The thresholds for all directions at each frequency were averaged to create a composite shaker audiogram for each of the spec ies (Fig. 3.4). The sharks had their most sensitive thresholds at the lowest freque ncies with increasing thresholds as the frequencies increased. The dipole audiogram for C. plagiosum yielded significantly lower thresholds than audiograms acquired fr om the shaker stimuli (p=0.018). At 20 Hz and 50 Hz the dipole particle acceleration m ean thresholds were more than 10 times lower than the particle acceleration threshol ds obtained with the shaker. A Tukey posthoc multiple comparisons test showed differenc es were statistically significant at 50 Hz (p=0.045) and 200 Hz (p=0.001), but not at 20 Hz and 100 Hz. DISCUSSION These directional hearing experiments are the first physiological measurements of directional hearing thre sholds in elasmobranch fishes. These results suggest that the ear of C. plagiosum is an omnidirectional particle ac celeration sensor (Kalmijn, 1988), as there were no significant differences among thres holds in each of the different directions (Fig. 3.3). These results are consistent with studies on hair cell polarities in elasmobranch fishes (Barber and Emerson, 1980; Corwin, 1981a). An examination of the winter skate, Raja ocellata showed a wide range of hair cell polarities depending on the endorgan (Barber and Emerson, 1980). The ut ricular macula had most cells in the anterior/posterior directions with some at varying degrees towards the dorsal/ventral

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89 directions. The saccular macula was predominan tly in the dorsal/ventral directions with a few cells in the anterior/posterior directions The lagenar macula showed varying angles towards the dorsal/ventral directions. It shoul d be noted that the ma cular sensory area of each endorgan is not typically flat, but more often curved and angled in specific directions. This is pa rticularly apparent in N. brevirostris in which the saccular macula was an S-shaped structure following the cont ours of the bottom of the saccule (Corwin, 1981a). Based on this distinct shape it appe ared that the hair cell polarizations of N. brevirostris cover all directions, which would contri bute to successful directional hearing abilities. The dipole hearing thresholds are significantly lower than the majority of other elasmobranchs (Banner, 1967; Kelly and Nelson, 1975; Casper a nd Mann, 2006; Casper and Mann, in press). This result suggests that sounds coming from above the shark should yield lower thresholds than othe r directions (previous monopole hearing experiments in elasmobranchs had sounds dire cted from the anterior). However, the whole-body acceleration data clearly show that there is no spec ific direction which yields consistently lower hearing thresholds than th e others (Fig. 3.3). The likely explanation for this involves the method of stimulation in each experiment. The directional hearing experiments use a shaker table to produce w hole-body accelerations of the sharks. As the shark’s body is being accelerated back and fort h, structures of greater density than the surrounding tissues, such as the otoconia, lag re lative to the rest of the shark body. This

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90 causes a shearing of the hair ce lls, thus stimulating the ear. This method of stimulation will only function as long as there is a density differential to create this lag. In the case of the macula neglecta, the hair cells are not mass-loaded with otoconia, but have a gelatinous cupula similar to the ha ir cells of the lateral line organs and semicircular canal cristae. This cupula likely would not be affect ed by the accelerations as its density is not large enough to create a lagging effect, and like the lateral line cupula, would need fluid flow in the posterior canal duct for movement to occur. Therefore, in the shaker experiments, it is highly likely that the sacculu s, utricle and lagena were being stimulated, but the macula neglecta was not. One of the conclusions drawn from the sh ark dipole hearing experiments (Casper and Mann, in press) is that the dipole stimulus creates a strong, localized velocity fluid flow from the vertical movement of the plastic ball. This fluid flow would be directed towards the parietal fossa, where it would cr eate a fluid flow in the posterior canal duct where the macula neglecta is located. Flui d flow within this canal across the cupula of the macula neglecta would cause a movement of the cupula, thereby shearing the hair cells and stimulating the endorgan. Based on th e significantly lower thresholds observed in the dipole experiments, it appears that th e macula neglecta is more sensitive than the other endorgans to localized flow (Fig. 3.4). However, if the macula neglecta is responding to particle velocity from fluid flow and the otoconia-based endorgans are responding to particle accelerations then th ere can’t be any dir ect comparison between

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91 thresholds. The thresholds from the vibra tional hearing experiment s (Fig. 3.4) are also closer to other monopole shark audiogra ms (Banner, 1967; Kelly and Nelson, 1975; Casper and Mann, 2006) suggesting that thes e experiments were only stimulating the otoconia. Similar directional hearing experime nt were conducted on the goldfish, Carassius auratus which has specialized Weberian ossi cles that transmit the sound pressure detected by the swim bladder as particle moti on to the inner ears. However, because the shaker table does not produce an appreciable sound pressure, C. auratus should be only exposed to particle motion putting it on a level “hearing” field as the sharks. Interestingly, C. auratus appears to have lower hearing thresholds at all frequencies, except 100 Hz, than the sharks even though th e swim bladder has been theoretically neutralized by the lack of sound pressure in the experiment. Two hypotheses for the lower thresholds could be mass loading by th e Weberian ossicles, and the composition of the otoliths in C. auratus versus the otoconia in elasmobran chs. The otoliths in teleosts are generally composed of a solid calc ium carbonate matrix, while elasmobranch otoconia are calcium carbonate, w ith exogenous siliceous material in a gelatinous matrix. It has been suggested that ear s with otoliths of a higher density are more sensitive to accelerations (Lychakov, 1990; Lychakov and Re bane, 2005). Therefore, the solid, dense otoliths of the C. auratus should result in a more sensitive ear than the less dense, gelatinous otoliths of sharks. Elasmobranch s can add to the density of their otoconia

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92 through the passive uptake of exogenous siliceous particles through the endolymphatic ducts (Stewart, 1906; Nishio, 1926; Fnge, 1982; Vilches-Troya et al., 1984; Hanson et al., 1990; Lychakov et al., 2000), but it is doubtful that they would be able to compensate enough to equal the acoustic abilit ies of a solid structure like a dense otolith. The hearing of C. auratus was measured in another shaker tabl e experiment (Fay, 1984) at 140 Hz with thresholds ranging from 7.74 x10-7 m/s2 for the most sensitive neurons to 7.74 x10-1 m/s2 for the least sensitive neurons. This ra nge falls about the da ta obtained in the current experiment for C. auratus evoked potentials at 100 Hz at 6.14 x10-3 m/s2. These experiments provide the first p hysiological evidence of elasmobranchs detecting sounds from all direct ions. Similar threshold were obtained at each of the directions tested which suggest s that the these sharks have omnidrectional ears, which is further supported by previous anatomical st udies on the inner ear hair cell polarities (Barber and Emerson, 1980; Corwin, 1981a). Composite audiograms obtained from the average of all seven directions shows that the C. auratus had lower thresholds than C. plagiosum and C. punctatum Based on the lower thresholds obtained from the dipole experiment with C. plagiosum it is likely that the directional shaker only stimulated the acceleration-sensitive otoconia end organs (saccu lus, utricle and lagena) of the inner ear and not the cupula-loaded macula neglecta, offering further evidence that the macula neglecta is most likely a velocity sensitive endorgan. These results are consistent with

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93 measurements showing that sharks are not as sensitive to sounds in the far-field, which would not likely stimulate the macu la neglecta (Casper and Mann, 2006).

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94 Figure 3.1 Diagram of the directional shaker table setup. Drawing not to scale.

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95

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96 Figure 3.2 A. Acceleration raw signals for a stimulus directed in the Z direction (up/down) as recorded from the three dimensional accelerometer. B. Auditory Evoked Potentials (AEPs) from the white-spotted bamboo shark, Chiloscyllium plagiosum in response to a 100 Hz signal at six signal levels. As the si gnal is decrease d in acceleration level (m/s2) the AEP signal also decreases until it is lost in the noise at 6.0-3 m/s2. C. 2048-point Fast Fourier Transfor m (FFT) of the same AEP for the shark in response to a 100 Hz sound. The arrow indicates the freque ncy doubling peak which occurs at 200 Hz.

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97 0 0.1 0.2 0.3 0.4 0.5 -0.5 0 0.5 Acceleration (m/s 2 ) 0 0.1 0.2 0.3 0.4 0.5 -0.5 0 0.5 0 0.1 0.2 0.3 0.4 0.5 -0.5 0 0.5 Time ( s ) Z Y X A

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98 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 Time ( s ) AEP Level (m/s2)1.7-1 8.6-2 4.3-2 2.2-2 1.1-2 6.0-3 B

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99 0 500 1000 1500 2000 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 Frequency (Hz)Evoked Potential ( V) C

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100 Figure 3.3 Directional hearing thresholds (m ean SE) for the white-spotted bamboo shark, Chiloscyllium plagiosum and the brown-banded bamboo shark, Chiloscyllium punctatum for each of the seven directions measur ed at A. 20 Hz, B. 50 Hz, C. 100 Hz, and D. 200 Hz. There was no significant differe nce between any of th e directions at any of the frequencies except at 50 Hz fo r interactions between the Z and 300 directions and the Z and 900 directions.

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101 20 Hz 1.00E-04 1.00E-03 1.00E-02 1.00E-010 degrees 30 degrees 60 degrees 90 degrees z x + z y + zParticle Acceleration (m/s2) White-Spotted Bamboo Shark Brown-Banded Bamboo Shark A

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102 50 Hz 1.00E-04 1.00E-03 1.00E-02 1.00E-010 degrees 30 degrees 60 degrees 90 degrees z x + z y + zParticle Acceleration (m/s2) White-Spotted Bamboo Shark Brown-Banded Bamboo Shark B

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103 100 Hz 1.00E-04 1.00E-03 1.00E-02 1.00E-010 degrees 30 degrees 60 degrees 90 degrees z x + z y + zParticle Acceleration (m/s2) White-Spotted Bamboo Shark Brown-Banded Bamboo Shark C

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104 200 Hz 1.00E-04 1.00E-03 1.00E-02 1.00E-010 degrees 30 degrees 60 degrees 90 degrees z x + z y + zParticle Acceleration (m/s2) White-Spotted Bamboo Shark Brown-Banded Bamboo Shark D

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105 Figure 3.4 Composite directional shaker audiogr ams (mean SE) of the white-spotted bamboo shark, Chiloscyllium plagiosum the brown-banded bamboo shark, Chiloscyllium punctatum and the goldfish, Carassius auratus These audiograms are compiled from the average of all of the thre sholds at each of the directions for each frequency tested. Also plotted is the dipole audiogram for C. plagiosum to compare responses from different stimuli. The dipole thresholds we re significantly lower than the directional shaker thresholds at 50 and 200 Hz for C. plagiosum

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106 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 050100150200Frequency (Hz)Particle Acceleration (m/s2) White-Spotted Bamboo Shark Brown-Banded Bamboo Shark Goldfish White-Spotted Bamboo Shark Dipole

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107 Chapter 4: The hearing thresholds of the Atlantic sharpnose shark, Rhizoprionodon terraenovae ABSTRACT The hearing thresholds of the Atlantic sharpnose shark, Rhizoprionodon terraenovae were measured in the field using auditory evoked potentials (AEP). The shark had most sensitive hearing at 20 Hz, the lowest freque ncy tested, with decreasing sensitivity at higher frequencies. Hearing thresholds we re lower than AEP thresholds previously measured for the nurse shark, Ginglymostoma cirratum and yellow stingray, Urobatis jamaicensis at frequencies below 200 Hz, a nd similar at 200 Hz and above. Rhizoprionodon terraenovae represents the closest comparison in terms of pelagic lifestyles to the sharks which have been obs erved in field attraction experiments. The sound pressure levels that would be equivalent to the particle accele ration thresholds of R. terraenovae were much higher than the sound leve ls in which closely related sharks were attracted suggesting a discrepancy betw een the hearing threshold experiments and the field attraction experiments.

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108 INTRODUCTION A wide range of experiments have been conducted to examine the hearing abilities of elasmobranch fishes. Field at traction experiments ha ve found that certain species of sharks are attracted to lower frequency (20-1000 Hz ), erratically pulsed sounds from distances up to several hundred mete rs (Nelson and Gruber, 1963; Richard, 1968; Myrberg et al., 1969; Nelson et al., 1969; Nelson and J ohnson, 1972; Myrberg et al., 1972; Myrberg 1978). The sounds played in these experiments we re likely at higher levels than most auditory stimuli that sh arks would be exposed to in their natural environments (Richard, 1968; Myrberg, 1978; Kalmijn, 1988). Also, the loudness of the sounds was recorded in reference to the sound pressure component of sound, which is not what sharks can hear. Only bony fishes w ith swimbladders are able to detect sound pressure, while all others, including elasmobr anchs, can only detect the particle motion component of sound (acceleration, velocity, di splacement). Without measuring acoustic particle motion it is not possibl e to characterize th e signals to which the sharks respond. The anatomy of the elasmobr anch inner ear is well stud ied (Tester et al. 1972; Corwin, 1977) and the pathways by which sound travels from the environment to the ear have been hypothesized (Tester et al. 1972; Fay et al., 1974; Corwin, 1977; Corwin, 1981A). Elasmobranchs have two proposed methods of detecting sound. 1) The first, the otolithic pathway, involves dire ct detection of the particle acceleration component of sound via the inner ear otoconia: the sacculus, utricle and lagena (though the lagena is

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109 primarily assumed to respond to angular acceler ations and not serve an acoustic purpose). The density of the shark’s body is approximate ly the same as the surrounding water. Therefore, sound travels through the body until it comes in contact with a structure of differing density. The otoconi a are denser than the surr ounding tissues and lag in response to the sound causing a shearing of th e sensory hair cells attached to these structures. This shearing causes stimulation of the hair cells and thus acoustic detection. The second method of detecting sound, the non-otolithic pathway, involves the fourth inner ear endorgan, the macula neglecta. The macula neglecta differs in that it has a cupula overlying the sensory hair cells rather than an otoconia and thus does not have a mass loaded structure of greater density for st imulation. It is believed that the macula neglecta is a particle velocity detector desi gned to detect sounds from above the shark’s head, based on the dorsal/ventr al polarization of the sens ory hair cells (Corwin, 1978; Corwin, 1981a; Corwin, 1983; Barber et al., 1985). The macula neglecta is located in the posterior canal duct in the dorsal portion of the ear just under an ar ea of loose connective tissue, the parietal fossa. As an animal sw ims over the top of the elasmobranch head, it creates a strong velocity flow which travel s through the parietal fossa and into the posterior canal duct via the fenestrae ovalis. This velocity flow cau ses the fluid in the posterior canal duct to move across the macu la neglecta, moving the cupula and shearing the sensory hair cells.

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110 Audiograms have also been acquired in se veral species of elasmobranchs (Kritzler and Wood, 1961; Olla, 1962; Banner, 1967; Nelson, 1967; Kelly and Nelson, 1975; Casper et al., 2003; Casper and Mann, 2006) though few were measured in terms of particle acceleration (Banner, 1967; Kelly and Nelson, 1975; Casper and Mann, 2006). Determining elasmobranch audiograms in te rms of sound pressure can indicate the frequency ranges that these species can detect, but it provides no data as to how well they can detect these sounds, if they do not detect sound pressure. Also, the majority of audiograms have been measured from deme rsal elasmobranchs, except for the lemon shark, Negaprion brevirostris (Banner, 1967). The majority of sharks which were observed in the field attraction experiments were pelagic. Thus, the lack of hearing data on these species makes it difficult to compar e elasmobranch hearing thresholds and the sound levels to which sharks were attracted. The goals of this study were to measure th e hearing thresholds of the piscivorous Atlantic sharpnose shark, Rhizoprionodon terraenovae and relate the thresholds to other elasmobranch audiograms. Several members of the same genus, R. porosus were observed in the attraction e xperiments (Richard, 1968; Myrber g et al. 1969). Therefore, this audiogram will provide a relevant comparison to the sound levels used in the field attractions experiments.

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111 MATERIALS AND METHODS Shark Hearing Thresholds Three juvenile Atla ntic sharpnose sharks, R. terraenovae (39-50 cm TL) were caught with ho ok and line off the beach at Little Gasparilla Island, Florida, USA. Upon capture each shark was quickly transported in a cooler to a dock area where the heari ng experiments were conducted. Hearing experiments followed the guidelines for th e care and use of animals approved by the Institutional Animal Care a nd Use Committee at University of South Florida protocol #2118. The auditory evoked potential (AEP) me thods described follow a similar protocol as previous elasmobranch AE P tests (Casper and Mann, 2006). Each subject was placed in stiff plastic mesh holders (2.54 cm x 2.54 cm holes). These holders were tightened with tie wrap s that were tight enough to keep the shark from moving, but did not affect breathing. Pieces of nylon rope were attached to either end of the plastic mesh and the shark was suspended in the water 2 m below a section of the dock (water depth = 3 m). The dock wa s 10 m from a mangrove fringed habitat. Bottom type was mud, with sparse sea grass. The water temperature was 32 C with a salinity of 34 ppt. The transducer (Aqua sonic Tactile Sound Underwater Speaker AQ339, Clark Synthesis, Littleton, CO USA) was hung with nylon rope from a different area of the dock 2.75 m from the shark’s head. Wire electrodes (12 mm length, 28 gauge lo w-profile needle electrode, Rochester Electro-Medical, Inc., Tampa, FL USA) were placed subdermally 1 cm posterior to the

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112 endolymphatic pores (recording electrode), in th e dorsal musculature 3 cm anterior to the dorsal fin (reference electrode), and free in th e water (ground electrode ). The electrodes were connected to a TDT pre-amplifier (HS 4, Tucker Davis Technologies, Gainesville, FL USA) which was then connected by a fi ber-optic cable to a TDT System II evoked potential workstation with TDT BioSig software. All sounds were pulsed tones that were 50 ms in duration and shaped with a Hanning window. Sounds above 20 Hz were de livered with a 70 ms presentation period (14/second), while 20 Hz sounds had a 1000 ms presentation period (1/second). Test frequencies ranged from 20-2000 Hz (20, 50, 100, 200, 300, 400, 800, 1000, 2000 Hz). Sounds were attenuated in 6 dB steps begi nning at the loudest le vel that could be generated at each frequency. The AEP wavefo rms were digitized at 25 kHz and averaged between 100-1000 times. More averages are n eeded as the signal moves closer to the threshold in order to pull the si gnal out of the AEP noise floor. A 2048-point Fast Fourier Transform (FFT ) was used to analyze the AEP signals in the frequency domain. The entire 70 ms window was FFT-transformed, because for many of the lower frequencies that were test ed the AEP signal took up the entire window. This was done at every frequency for the anal ysis to remain consistent. An AEP was determined to be present if the recorded signa l showed a doubling of the sound frequency (e.g. a 400 Hz peak when the signal played wa s 200 Hz) with a peak at least 3 dB above the AEP noise floor. The AEP noise floor is estimated from the AEP power spectrum

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113 with a window of 100 Hz around the doubling frequency (i.e. 50 Hz on each side of the peak). This frequency doubling occurs in all low frequency fish AEP testing (Mann et al., 2001; Egner and Mann, 2005; Casper and Mann, 2006). Upon completion of the experiment, each sh ark was measured and released. For calibration a pressure/velocity probe (Uniaxia l Pressure/Velocity Probe, Applied Physical Sciences Corporation, Groton, CT USA) was positioned in the same location where the head of the shark had been. The probe contained a velocity geophone (sensitivity 212 mV/cm/s, bandwidth 10 Hz-1 kHz) and a hydrophone (sensitivity: -176 dB re 1 V/Pa, bandwidth 10 Hz-2 kHz), which could simulta neously record sound pressure and particle velocity (Table 1). Calibration with the ge ophone was performed in all orientations (0 horizontal (X-axis), 90 horizon tal (Y-axis), and vertical (Z -axis)) and all calibrations were computed as the Root Mean Square (RMS) for the magnitude of the three axes combined. The hydrophone was omni-directiona l and therefore did not need to be measured along different axes. Many research ers have suggested th at the inner ear of fishes act as an accelerometer and theref ore detect acoustic particle acceleration (Kalmijn, 1988; Fay and Edds-Walton, 1997; Braun et al., 2002; Bass and McKibben, 2003). Therefore, all audiogram s have hearing thresholds sh own in units of particle acceleration (m/s2). Particle velocity of tonal si gnals can be converted to acceleration with the following equation: ac celeration = velocity x (2 x x frequency). The

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114 acceleration thresholds are also given as a f unction of the magnitude of the three (X, Y, Z) directions measured. RESULTS Shark Hearing Thresholds. Rhizoprionodon terraenovae AEPs showed frequency doubling where the frequency of the AEP was ab out twice the stimulus frequency as seen in previous AEP elasmobranch studies (Casper and Mann, 2006). The R. terraenovae audiogram had a similar shape as other elas mobranch audiograms with most sensitive hearing at low frequencies and increasing thresholds with in creasing frequency (Fig 4.1). Ambient noise recordings were consistently around 1 x 10-3 m/s2 (Fig. 4.1) DISCUSSION The audiogram for R. terraenovae is the second audiogram recorded from the family Carcharhinidae in terms of particle motion. The first was determined by Banner (1967) for N. brevirostris (Fig. 4.1). Kritzler and Wood (1961) measured the hearing thresholds of the bull shark, Carcharhinus leucas using only a pressure hydrophone, for which is it not possible to determine the par ticle motion. Responses ranged from 100 Hz to 1400 Hz referenced to an unspecified noise level. The R. terraenovae audiogram shared a similar shape and frequency respons e with other elasmobranch audiograms that

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115 were obtained using AEP me thods at 200 Hz and above (Casper and Mann, 2006) (Fig. 4.2). Below 200 Hz R. terraenovae had lower thresholds than previously measured AEP audiograms. However, the results are diffe rent from audiograms obt ained using classical conditioning methods (Banner, 1967; Kelly and Nelson, 1975 ). Rhizoprionodon terraenovae has lower thresholds than the horn shark, Heterodontus francisi (Kelly and Nelson, 1975) at all frequencies tested, alt hough it should be noted that many of the lower frequencies tested in th e horn shark were likely masked by ambient noise levels (Fig. 4.2). Negaprion brevirostris had lower thresholds than R. terraenovae at 20 Hz, but higher thresholds at all other frequencies tested (Banner, 1967). Previous research has suggested that the macula neglecta inner ear endorgan is a low frequency particle velocity detector in elasmobranchs (Casper and Mann, in press). Corwin (1978) found that the silky shark, Carcharhinus falciformis (reported species menisorrah ), had a significantly larger macula ne glecta with a greater number of sensory hair cells than any other species of elasmobr anchs. suggesting enhanced hearing abilities for more active, piscivorous elasmobranchs. If this pattern holds for R. terraenovae then this could help to explain the lower threshol ds observed in this species at 100 Hz, and presumably lower frequencies, compared to demersal elasmobranchs such as the nurse shark, Ginglymostoma cirratum and yellow stingray, Urobatis jamaicensis which also have had hearing measured with AEPs (Casper and Mann, 2006) and H. francisi using classical conditioning methods (Fig. 4.2). However, it is not clear whether simply

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116 increasing the number of hair cells will increase sensitivity. Another possible explanation for the differences is that the test location for R. terraenovae was in a different acoustic environment than the G. cirratum and U. jamaicensis which were tested in a shallow cement lagoon (1.05 m) with a curved bottom. Rhizoprionodon terraenovae was tested in deeper water (3 m) w ith a soft, muddy bottom. It should also be noted that the ambient noise levels in the R. terraenovae experiment were about three orders of magnitude higher than the nurs e shark and stingray experiments (~1.0 x 10-3 m/s2 versus ~1.0 x 10-6 m/s2), though this did not appear to affect the hearing thresholds as R. terraenovae had lower thresholds (Fig. 4.1). Using the equation: p= cv where, p=pressure (Pa) =density of medium (1030 kg/m3) c=speed of sound in the medium (1500 m/s) v= particle velocity (m/s) the equivalent far-field sound pressure levels that would be associated with a given particle velocity level (determined from the particle acceleration values) can be estimated for the R. terraenovae thresholds (Fig. 4.3). These fa r-field pressure-based threshold estimates of R. terraenovae (Fig. 4.6) can be compared to the sound levels which were

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117 produced during the field at traction studies in which R. porosus was observed (Richard, 1968; Myrberg et al., 1969). Based on the sound pressure thresholds of R. terraenovae it appears that the sound levels from the fiel d attraction experiment s were well below the hearing thresholds of the sp ecies (Fig. 4.3). A similar di screpancy was observed between measured hearing thresholds and the reported sound levels in which sharks were observed to be attracted to in a previous experiment with G. cirratum (Casper et al., 2006). There are several possible explanations for these observed discrepancies with R. terraenovae It has been suggested that AEP measurements can underestimate hearing thresholds in fishes compared to behavioral training methods (Mann et al., 2001). This could help to explain the lo wer thresholds observed in N. brevirostris compared to R. terraenovae Another possible explan ation could be other stimu li could have attracted sharks to the testing locations. In many of the test trials in one of the experiments (Richard, 1968) sharks appeared to be following other fishes into the test area. Several of these fishes, including snapper and grouper, have swimbladders and likely can detect lower level sounds than R. terraenovae. It has also been observed in many of the field attraction experiments that the sharks responded erratically wi th agitated behaviors close to the speakers, which was likely due to the strong electromagnetic fields being produced by these speakers. This would provide anot her strong visual stimulus to which other sharks could respond from greater distances.

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118 Future experiments could attempt to r ecreate the field attrac tion studies in an attempt to remove external stimuli which could also result in the attraction of the sharks. The underwater speaker can be shielded us ing a faraday cage which would remove any electromagnetic field being produced doing the sound production. This electromagnetic field is likely what caused the sharks to be have erratically as they approached the speaker. A second modification to the experi ments would be to conduct them at night. This would presumably remove any visual stimul us that sharks outside of the testing area could use to follow other sharks ’ behaviors to the testing area. The movement patterns of the sharks could still be monitored using satel lite or other tracking means. It would also be useful to measure the sound field create d by the underwater speakers using a particle motion sensitive sensor as this is the component of sound that sharks de tect. If the sharks are still observed in the testi ng area then this would provide further evidence that sharks are attracted by thes e underwater sounds.

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119 Table 4.1 Example of the sound pressure levels measured by the pressure/velocity probe and the associated particle acc eleration levels that were converted from the recorded particle velocities. These le vels represent the loudest sounds which were produced by the underwater transducer at each frequency.

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120 Frequency (Hz) Sound Pressure (dB re 1Pa) Particle Acceleration (m/s2) 20 83.3 1.32 x 10-3 50 115.3 2.31 x 10-3 100 112.9 3.72 x 10-3 200 111.5 9.80 x 10-3 300 108.3 6.38 x 10-3 400 117.7 1.93 x 10-2 500 115.4 1.32 x 10-2 600 101.3 1.04 x 10-2 800 110.0 3.29 x 10-2 1000 120.5 3.03 x 10-2

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121 Figure 4.1 Audiogram (mean SE) of the Atlantic sharpnose shark, Rhizoprionodon terraenovae plotted against the ambient noise levels which were present during the hearing tests.

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122 0.0001 0.001 0.01 0.1 02004006008001000Frequency (Hz)Particle Acceleration (m/s2) Ambient Noise Atlantic Sharpnose Shark

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123 Figure 4.2 Particle acceleration au diograms of all elasmobranchs in response to a monopole sound stimulus (underwater speaker) The open shapes are elasmobranch audiograms obtained using auditory evoked pot entials (AEP) and the filled shapes are audiograms obtained using clas sical conditioning methods. Standard error lines are present when available.

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124 0.0001 0.001 0.01 0.1 101001000Frequency (Hz)Particle Acceleration (m/s2) Nurse Shark (Casper and Mann 2006) Yellow Stingray (Casper and Mann 2006) Lemon Shark (Banner 1967) Horn Shark (Kelly and Nelson 1975) Atlantic Sharpnose Shark

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125 Figure 4.3 The sound pressure needed to produce part icle accelerations equivalent to the Atlantic sharpnose shark, Rhizoprionodon terraenovae audiogram in a plane propagating wave (square symbols). The sound pressure levels used in two field attraction experiments in which members of the Rhizoprionodon genus were attracted are plotted for comparison. Distances from the sound sour ce to the hydrophone for measurements of SPL were 1m for Richard (1968) and the R. terraenovae experiment, while they were made at 18.5 m for Myrberg et al. (1969). Sound pressure audiograms for the nurse sharks, Ginglymostoma cirratum are calculated from the recorded velocities using the equation P= cv (where P=pressure (Pa), =density of the medium (1030 kg/m3), c=speed of sound in medium (1500 m/s), v=velocity (m/s)). The pressures were then log transformed to convert to s ound pressure levels (dB re 1 Pa). The sound levels are based on RMS levels. Richard (1968) and My rberg et al. (1969) sound levels are based on spectrum levels.

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126 100 105 110 115 120 125 130 135 140 145 150 02004006008001000Frequency (Hz)Sound Pressure Level (dB re 1 Pa) Atlantic Sharpnose Shark Audiogram Richard 1968 Myrberg et al. 1969

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127 CONCLUSION Elasmobranch Audiograms The goal of this dissertation was to build upon existing knowledge of elasmobranch hearing. The results yielded he aring measurements which are in conflict with the intensities of sounds th at resulted in the attraction of sharks in the field (Nelson and Gruber, 1963; Richard, 1968; Myrberg et al., 1969; Nelson et al., 1969; Nelson and Johnson, 1972, 1976; Myrberg et al., 1972; Myrb erg et al., 1976, 1978; Myrberg 1978). Some researchers have sugge sted that AEP measurements can overestimate hearing thresholds in fishes (Mann et al., 2001), but Casper et al. (2 003) found no significant difference in hearing thres holds of the little skate, Raja erinacea using both behavioral and AEP methods. Furthermore, behavioral audiograms of elasmobranchs (Banner, 1967; Kelly and Nelson, 1975) yielded similar th resholds as the AEP audiograms (Casper et al. 2006). This elasmobranch hearing data combined with the lack of any anatomical hearing specializations results in confoundi ng discrepancies between the earlier field attraction experiments and the curren t hearing threshold data. Elasmobranch Mechanisms of Sound Detection Elasmobranchs lack a swimbladder or a ny other kind of hear ing specialization that would suggest sensitive he aring or an ability to detect the sound pressure component of sound. It is likely that th eir primary means of detecti ng sound involves the use of the

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128 otoconia to detect particle motion. The macula ne glecta is also believed to be involved in acoustic detection. Prior to this dissertati on, the few audiograms which had been acquired suggested a relatively narrow frequency range (20-1 400 Hz) with most sensitive hearing at low frequencies a nd increasing thresholds with increasing frequency. Acoustic particle motion is higher close to the sound source, or in the near field, and then falls off fairly quickly with distance, into the fa r field. This depends on the type of sound stimulus as monopoles tend to propagate fart her than more complex stimuli such as dipoles or quadrupoles. Sound propagation is also frequency dependent. The near field of a sound stimulus is generally considered to be a distance of one wavelength, so a low frequency sound would have a larger near field than a high frequency sound. Based on the frequency range this woul d create a near field of up to 75 m for a 20 Hz signal and about 1 m for a 1400 Hz signal. This research indicates that elasmobranchs are not designed for sensitive long-distance hearing. Audiograms were obtained in several species of sharks and rays, including the nurse shark, Ginglymostoma cirratum and Atlantic sharpnose shark, Rhizoprionodon terraenovae Their audiograms had best sensitiviti es at low frequencies and increasing thresholds with increasing frequency, as had been previously found for other elasmobranches (Banner, 1967; Kelly a nd Nelson, 1975). However, the highest frequency at which a response could be obtained was only 1000 Hz, less than the 1400 Hz response observed in the bull shark Carcharhinus leucas (Kritzler and Wood, 1961).

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129 In response to a monopole stimulus, R. terraenovae displayed lower thresholds at frequencies below 200 Hz than any ot her shark except for the lemon shark, Negaprion brevirostris (Banner, 1967). At 200 Hz and above all of the elasmobranchs tested yielded similar thresholds. The horn shark Heterodontus francisi and white-spotted bamboo shark Chiloscyllium plagiosum demonstrated differing results with a dipole source. These sharks only responded up to 300 Hz and both species were found to have thresholds well below any of the species te sted with a monopole stimulus. At 20 Hz these sharks responded with a similar threshol d as the lemon shark, previously considered to have the lowest threshold for any elasmobr anch. These dipole experiments suggest that the macula neglecta was likely being stimulat ed from the strong particle velocity flow being created by the dipole stimulus directly above the head of the shark. The monopole source, located in front of the shark’ s body, was apparently stimulating the three otoconial endorgans with less stimulation of th e macula neglecta. This stimulation of the endorgans was supported by moving the dipole st imulus over the shark’s dorsal surface. It was found that when the dipole was positi oned above the parietal fossa, a region long suspected of being a pathway of sound trav el to the inner ear, the strongest evoked potentials were produced. These results sugge st that the macula neglecta is likely a velocity detector, compared with the otoconia which are acc eleration detectors. For stimuli close to the shark head (<10 cm), the macula neglecta response dominates the evoked potential. Being able to detect a ve locity flow from above the elasmobranch,

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130 such as from a swimming fish, would be usef ul for demersal sit-a nd-wait predators which must remain motionless, and often buried in the sand while waiting for prey items to approach. Reconciling Field Attraction Experime nts: Suggestions for future research The current work has provided evidence that while elasmobranchs can detect sounds, they do not have sensitive hearing compar ed to bony fishes or the ability to detect most natural sounds they encounter in the far field. The data suggest that sharks hear sound primarily in the near field where the part icle motion of the stimulus is relatively high. Furthermore, sharks would have diffi culty detecting many of the sounds generated in previous attraction studies, even at the exceptionally loud le vels. However, it should be pointed out that all species that have had their hearing thresholds measured are demersal and/or coastal sharks and many of the species that were observed in the field attraction experiments were predominantly pela gic sharks. Corwin (1978) suggested that pelagic sharks have large, more develope d macula neglectas compared to demersal species which could yield better hearing abilities. It is likely that the sharks in the field attraction studies were using a combination of several sensory modalities working in unison that led the sharks to the study locations. Almost every field attraction experiment ci ted shark behavior cl ose to the underwater speakers as “fast swimming motions” and “erra tic” (Richard, 1968; Myrberg et al., 1969;

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131 Nelson et al., 1969; Nelson and Johnson, 1972, 1976; Myrberg et al., 1972; Myrberg et al., 1976, 1978; Myrberg 1978). The speakers lik ely also produced an electromagnetic field that may have been detected by the shar ks. Furthermore, since these studies were performed in clear water, the exaggerated swimming behaviors of sharks close to the speaker could visually attrac t other sharks from larger distances. Nelson and Johnson (1972) found that as many sharks appeared after the sound was turned off during the control period as showed up during the testing period (Nelson and Johnson, 1972). Another experiment monitored teleosts as well as sharks attracted to underwater sounds, and in many of their trials sharks would appe ar to be following other fishes to the speaker location (Richard, 1968). It is likely that these fishes (sna ppers and groupers) have more sensitive hearing than sharks due to the pres ence of a swimbladder and could detect these sounds from farther distances. It remains unc lear as to what degree sound played a role in the acoustic attraction experiments, but it appears likely that it was not the only stimulus present to which the sharks could react. This discrepancy could be resolved in future field attraction experiments that control for external non-acoustic stimuli. The underwater speaker can be shielded using a Faraday cage which would remove any el ectromagnetic field produced during sound production. This electromagnetic field could have caused the sh arks to behave erratically as they approached the speaker. A second m odification to the expe riments would be to conduct them at night. This would presumably remove any visual stimulus that sharks

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132 outside of the testing area could use to follow other sharks’ behaviors to the testing area. The movement patterns of the sharks could st ill be monitored using acoustic tracking. It would also be useful to measure the sound field created by the unde rwater speakers using a particle motion sensitive sensor as this is the component of sound that sharks detect. Testing the hearing thresholds of pelagic sharks which were obs erved in the field attraction experiments is also imperative to be able to draw valid conclusions about the ability for these sharks to be able to detect the sounds which were being broadcasted. If the sharks are attracted with these controls, th en this would provide further evidence that sharks are attracted by these underwater sounds.

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133 Figure C.1 All existing audiograms for elasm obranchs in response to particle motion. The black shaded symbol audiograms represen t the classical conditioning studies with the lemon shark (Banner, 1967) and the horn shark (Kelly and Nelson, 1975). The white symbol audiograms represent AEP audiograms us ing a monopole speaker. The dark grey shaded symbols represent AEP audiograms us ing a dipole stimulus. The light grey shaded audiograms represent the di rectional shaker AEP audiograms.

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134 0.00001 0.0001 0.001 0.01 0.1 101001000Frequency (Hz)Particle Acceleration (m/s2) Nurse Shark Yellow Stingray Lemon Shark (Banner 1967) Horn Shark (Kelly and Nelson 1975) Horn Shark Dipole White-Spotted Bamboo Shark Dipole White-Spotted Bamboo Shark Shaker Brown-Banded Bamboo Shark Shaker Atlantic Sharpnose Shark

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135 REFERENCES Abboud, J.A. & Coombs, S. (2000). Mechanosensory-based or ientation to elevated prey by a benthic fish. Mar. Fresh. Behav. Physiol. 33, 261-279. Astrup, J. and Mhl, B. (1993). Detection of inte nse ultrasound by the cod, Gadus morhua J. Exp. Biol. 182 71-80. Astrup, J. and Mhl, B. (1998). Discrimination between high and low repetition rates of ultrasonic pulses by the cod. J. Fish Biol. 52 205-208. Banner, A. (1967). Evidence of sensitivity to acoustic displacements in the lemon shark, Negaprion brevirostris (Poey). In Lateral Line Detectors (ed. P.H. Cahn), pp 265273. Bloomington :Indiana University Press. Banner, A. (1968). Attraction of young lemon sharks, Negaprion brevirostris to sound. Copeia 4 871-872. Banner, A. (1972). Use of sound in predation by young lemon sharks, Negaprion brevirostris (Poey). Bull. Mar. Sci. 22 251-283. Barber, V.C. and Emerson, C.J. (1980). Scanning electron mi croscopic observations on the inner ear of the skate, Raja ocellata Cell Tissue Res. 205 199-215. Barber, V.C., Yake, K.I., Clark, V.F. and Pungur, J. (1985). Quantitative analyses of sex and size differences in the macula ne glecta and ramus negl ectus in the inner ear of the skate, Raja ocellata Cell Tissue Res. 241 597-605. Barry, M.A. (1987). Afferent and effere nt connections of the pr imary octaval nuclei in

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136 the clearnose skate, Raja eglanteria J. Comp. Neurol. 266 457-477. Bass, A.H. and Clark, C.W. (2003). The physical acoustics of underwater sound communication. In Acoustic Communication (ed. A.M., Simmons, A.N. Popper and R.R. Fay), pp 15-64. New York:Springer-Verlag,. Bass, A.H. and McKibben, J.R. (2003). Neural mechanisms and behaviors for acoustic communication in teleost fish. Prog. Neurobiol. 69 1-26. Batteau, D.W. (1967). The role of the pi nna in human localization. Proc. R. Soc. Lond. 168B 158-180. Bleckmann, H., Bullock, T.H. and Jorgensen, J.M. (1987). The lateral line mechanoreceptive mesencephalic, diencephalic, and telencephalic regions in the thornback ray, Platyrhinoidis triseriata (Elasmobranchii). J. Comp. Physiol. 161A 67-84. Bleckmann, H., Weiss, O. and Bullock, T.H. (1989). Physiology of lateral line mechanoreceptive regions in the elasmobranch brain. J. Comp. Physiol. 164A 459-474. Braun. C.B. and Coombs, S. (2000). The overlapping roles of the inner ear and lateral line: the active space of dipole source detection. Phil. Trans. R. Soc. Lond. 355B 1115-1119. Braun, C.B., Coombs, S. and Fay, R.R. (2002). What is the nature of multisensory interaction between octavol ateralis sub-systems? Brain Behav. Evol. 59 162-176.

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ABOUT THE AUTHOR Brandon Casper graduated with a Bachel or of Science degree in Biology from Ohio University in 1999. He then moved on to Woods Hole, MA where he accomplished his Master of Arts degree in Marine Biology in 2001 with the the thesis titled: The Hearing Abilities of the Little Skate, Raja erinacea : A Comparison of Two Methods. Following this, he took a year off from school to work as a Research Assistant for Dr. David Mann at the College of Marine Science at University of South Florida. He decided to continue his education in this lab by working towards his PhD in Biological Oceanography. While working on this PhD, he received several awards including the American Elasmobranch Society Donald R. Nelson Research Award as well as an American Elasmobranch Society Travel Grant to attend a conference. He also received the Tampa Bay Parrot Head Fellowship and Ri ggs Endowed Fellowship from the College of Marine Science. The results of his re search have been published in several peerreviewed journals.


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ABSTRACT: The hearing abilities of elasmobranch fishes were examined in response to several types of stimuli using auditory evoked potentials (AEP). Audiograms were acquired for the nurse shark, Ginglymostoma cirratum, the yellow stingray, Urobatis jamaicensis, in a controlled environment using a monopole underwater speaker. A dipole stimulus was used to measure the hearing thresholds of the horn shark, Heterodontus francisi, and the white-spotted bamboo shark, Chiloscyllium plagiosum. The dipole experiments yielded much lower thresholds than any other experiment, suggesting that this type of sound specifically stimulated the macula neglecta by creating a strong velocity flow above the head of the shark. A shaker table was created to measure the directional hearing thresholds of the C. plagiosum and the brown-banded bamboo shark, C. punctatum. This experiment showed that these sharks could sense accelerations equally in all directions suggesting that they have omnidirectional ears. The results also yielded higher thresholds than with the dipole, suggesting that the macula neglecta was not stimulated as the sharks were being accelerated. An audiogram was also acquired for the Atlantic sharpnose shark, Rhizoprionodon terraenovae, using a monopole speaker in the field. This experiment revealed that the hearing thresholds did not appear to be masked by ambient noise levels, and resulting thresholds yielded the lowest levels detected by any elasmobranch using AEPs. Taken together, these experiments show that sharks are most sensitive to low frequency sounds in the near field and use both their otoconial endorgans as well as the macula neglecta to sense particle motion.
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