Tactile Abilities of the Florida Manatee (Trichechus manatus latirostris)

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Tactile Abilities of the Florida Manatee (Trichechus manatus latirostris)
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english
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Gaspard, Joseph C
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University of Florida
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Veterinary Medical Sciences, Veterinary Medicine
Committee Chair:
Reep, Roger L
Committee Members:
Francis-Floyd, Ruth
Mcguire, Peter M
Lefebvre, Lynn Walsh
Bauer, Gordon B
Mann, David A

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Subjects / Keywords:
manatee -- sirenian -- tactile -- vibrissae -- vibrotactile
Veterinary Medicine -- Dissertations, Academic -- UF
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Veterinary Medical Sciences thesis, Ph.D.
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theses   ( marcgt )
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Abstract:
Manateesinhabit the coastal and inland waters of Florida.  They seem to have little difficultynavigating in turbid waterways and maneuvering around underwater obstacles.  Previous research has demonstrated their goodhearing and noise localization abilities; however their visual acuity is quitepoor.  Manatees possess follicle sinuscomplexes (FSCs) over their entire body, and anatomical and behavioral evidencesuggests that FSCs form a sensory array system for detecting hydrodynamicstimuli analogous to the lateral line system of fish.  The FSCs were tested in a series ofexperiments to assess the sensitivity of the facial and post-facial vibrissaeto hydrodynamic stimuli through threshold and directionality assessment.  Among other findings, the results of thisresearch demonstrated the manatee’s ability to detect particle displacementdown to a nanometer.  This is consistentwith anatomical and behavioral evidence that manatees are tactile specialists,evidenced by their specialized facial morphology and the use of these vibrissaeduring feeding and the active investigation/manipulation of objects.
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In the series University of Florida Digital Collections.
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Includes vita.
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by Joseph C Gaspard.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
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Adviser: Reep, Roger L.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-05-31

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lcc - LD1780 2013
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UFE0045261:00001


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TACTILE ABILITIES OF THE FLORIDA MANATEE ( TRI CHECHUS MANATUS LATIROSTRIS) By JOSEPH CHARLES GASPARD III A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013 1

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2013 Joseph Charles Gaspard III 2

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To my family, who is always there for me with their love, support, and inspiration 3

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ACK NOWLEDGMENTS I thank my wife, Teresa, and my childr en, Greer and Laken, for giving me the strength and support to achieve my goals. You have enriched my life more than I could ever put into words. Everything I do is for you I love you. I would like to thank my parents for supporting me and allowing me to follow my dream. I would like to thank Dr. Roger Reep, my advisor, who has provided me with an altruistic view of what it is we do as sci entists. Without your patience and guidance, I would have stumbled through this journey and may not have gotten up. I would like to thank Dr. Gordon Bauer and Dr. David Mann for mentoring me for many years and helping me grow in this field. I would like to thank my Committee Members, Drs. Peter McGuire, Lynn Lefebvre, Ruth Francis-Floyd, and Don Samuelson, for their invaluable questions, insights, and knowledge that helped mold my graduate career and my research. I would like to thank the st aff at Mote Marine Laboratory & Aquarium, especially Katharine Nicolaise n, Laura Denum, Kimberly Dziuk, and LaToshia Read, for helping me conduct the research as well as MML for supporting my research. I would like to thank Dr. Alex Costidis for embarking on this academic rollercoaster with me and the regular intellectual banter battles. I would like to thank Dr. Debborah Colbert for taking a chance on an intern, giving me an oppor tunity to work in this field, and mentoring me during the beginning of my profe ssional career. I would like to thank the University of Florida for allowing me to be part of an amazing institution, and especially Sally OConnell who held my hand throughout this entire process. Thank you. I would like to thank Ronnie and John Enander and the Thurell Family for supporting the research and their undy ing excitement about the work. 4

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The research was permitted by the United St ates Fish and Wildlife Service (Permit MA837923). This work was supported by the National Science Foundation (IOS0920022/0919975/ 0920117). All experi mental procedur es were approved by the Mote Marine Laboratory IACUC pr ior to implementation. 5

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TABL E OF CONTENTS page ACKNOWLEDG MENTS..................................................................................................4 LIST OF TABLES............................................................................................................8 LIST OF FI GURES..........................................................................................................9 ABSTRACT ...................................................................................................................11 1 INTRODUC TION....................................................................................................12 Hair in Mammals .....................................................................................................12 Manatee Hair and Behavi or....................................................................................13 Vibrissae Comparative Distr ibution and Innervation............................................14 Vibrissae Compar ative Func tion...........................................................................15 Vibrissae in Manatees A Mammalian Later al Li ne?.............................................15 Project Obje ctives...................................................................................................19 Chapter 2 Ob jective..........................................................................................19 Chapter 3 Ob jective..........................................................................................19 Chapter 4 Ob jective..........................................................................................19 Significance of the Proj ect......................................................................................19 2 DETECTION OF HYDRODYNAMIC STIM ULI BY THE FACIAL VIBRISSAE OF THE FLORIDA MANATEE ( TRICHECHUS MANA TUS LATIROSTRIS )................20 Backgroun d.............................................................................................................20 Materials and Methods............................................................................................23 Subjec ts............................................................................................................23 Experiment I Tactogr am................................................................................24 Procedur es.................................................................................................24 Equipm ent..................................................................................................25 Experiment II Rest riction Te sts......................................................................28 Experiment III Si gnal Detect ion.....................................................................28 Result s....................................................................................................................29 Experiment I Tactogr am................................................................................29 Experiment II Rest riction Te sts......................................................................30 Experiment III Si gnal Detect ion.....................................................................30 Discussio n ..............................................................................................................31 3 DETECTION OF HYDRODYNAMIC STIMULI BY THE POST-FACIAL VIBRISSAE OF THE FLORIDA MANATEE ( TRICHECHUS MANATUS LATIROSTRIS).......................................................................................................48 Backgroun d.............................................................................................................48 Materials and Methods............................................................................................50 6

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Subjec ts............................................................................................................ 50 Experiment I Tactogr am................................................................................50 Procedur es.................................................................................................50 Equipm ent..................................................................................................52 Experiment II Rest riction Te sts......................................................................55 Result s....................................................................................................................56 Experiment I Tactogr am................................................................................56 Experiment II Rest riction Te sts......................................................................56 Discussio n..............................................................................................................57 4 DETECTION OF DIRECTIONALITY OF HYDRODYNAMIC STIMULI BY THE POST-FACIAL VIBRISSAE OF THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS ).....................................................................................79 Backgroun d.............................................................................................................79 Materials and Methods............................................................................................79 Subjec ts............................................................................................................79 Procedur es.......................................................................................................80 Equipm ent........................................................................................................ 81 Result s....................................................................................................................84 Discussio n..............................................................................................................84 5 CONCLUS ION........................................................................................................89 Signific ance............................................................................................................89 LIST OF RE FERENCES...............................................................................................91 BIOGRAPHICAL SKETCH ............................................................................................97 7

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LIST OF TABLES Table page 1-1 Weber Fracti ons.................................................................................................18 2-1 Mesh netti ng.......................................................................................................35 2-2 Facial threshold va lues and false alarm rate......................................................36 2-3 Restricted facial vi brissae thres hold valu es........................................................37 2-4 Signal Dete ction Th eory.....................................................................................38 3-1 Post-facial th reshold values................................................................................60 3-2 Post-facial threshold values fo r the right-side front loca tion...............................61 3-3 Post-facial threshold values for the right-side mid location.................................62 3-4 Post-facial threshold values for the right-side rear loca tion................................63 3-5 Post-facial threshold values for the left-side front loca tion..................................64 3-6 Restricted post-facial vibrissae thres hold val ues................................................65 4-1 Directionality test per centages ............................................................................87 8

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LIST OF FIGURES Figure page 2-1 Correct response................................................................................................39 2-2 Manatee stationed for facial vibrissae testing.....................................................40 2-3 Experimental setup for fa cial vibriss ae testin g....................................................41 2-4 Manatee stationed for restricted facial vibriss ae testin g.....................................42 2-5 Threshold values for faci al vibrissae Displacem ent..........................................43 2-6 Threshold values for faci al vibrissae Veloci ty...................................................44 2-7 Threshold values for facial vibrissae A ccelerati on............................................45 2-8 Signal detecti on for Buffett.................................................................................46 2-9 Signal detec tion for Hugh...................................................................................47 3-1 Manatee stationed for post-fa cial vibriss ae testin g.............................................66 3-2 Four testing locations for pos t-facial vibris sae tactogram...................................67 3-3 Shaker set-up with waterproof housing ..............................................................68 3-4 Manatee weari ng neoprene wrap.......................................................................69 3-5 Manatee stationed for restricted po st-facial vibri ssae test ing.............................70 3-6 Threshold values for post-fa cial vibrissae Displacement..................................71 3-7 Threshold values for post-fa cial vibrissae Velocity...........................................72 3-8 Threshold values for post-faci al vibrissae Accelera tion....................................73 3-9 Threshold values for restricted pos t-facial vibriss ae Displac ement..................74 3-10 Threshold values for restricted post-facial vibri ssae Velocity...........................75 3-11 Threshold values for restricted post-facial vibrissae Accelera tion....................76 3-12 Comparison of threshold values for displacement detec tion by Buffett..............77 3-13 Comparison of threshold values for displacement detection by Hugh................78 4-1 Manatee stationed for directionalit y detection testi ng.........................................88 9

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LIST OF ABBREVIAT IONS BLH bristle like hair f frequency FA false alarm FSC follicle sinus complex MML Mote Marine Laboratory & Aquarium USFWS United States Fish and Wildlife Service 10

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11 Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy TACTILE ABILITIES OF THE FLORIDA MANATEE ( TRICHECHUS MANATUS LATIROSTRIS) By Joseph C. Gaspard III May 2013 Chair: Roger L. Reep Major: Veterinary Medical Sciences Manatees inhabit the coastal and inland wate rs of Florida. They seem to have little difficulty navigating in turbid waterways and maneuvering around underwater obstacles. Previous research has demonstrated their good hearing and noise localization abilities; however their visual acuity is quite poor. Manatees possess follicle sinus complexes (FSCs) over their ent ire body, and anatomical and behavioral evidence suggests that FSCs form a sensory arra y system for detecting hydrodynamic stimuli analogous to the lateral line syst em of fish. The FSCs we re tested in a series of experiments to assess the sensitivity of the facial and post-facial vibrissae to hydrodynamic stimuli through threshold and directionality assessment. Among other findings, the results of this research demonstrated the manatees ability to detect particle displacement down to a nanometer. This is consistent with anatomical and behavioral evidence that manatees are tactile specialists, evidenced by their specialized facial morphology and the use of these vibrissae during feeding and the active investigation/manipulat ion of objects.

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CHA PTER 1 INTRODUCTION Hair in Mammals Mammals are warm-blooded, air-breathing vertebrates that give birth to live young who then nurse from their milk-producing mothers. An important characteristic of all mammals is the presence of hair. Hair is very apparent on most mammalian species, particularly the terrestrials. T he mane of a lion or the pelage of a grizzly bear easily identifies each as a member of the Mammalian class. It ma y be difficult to visualize, but even the bottlenose dolphin, w hose body has evolved for life in an aquatic environment, has or had hair at some point duri ng its life history. The manatees closest living relative, the dugong, also possesses body hairs that differ anatomically from those of the manatee, and have yet to be fu lly studied (J. Lanyon, pers. comm.). Hair itself can play a number of role s depending on the ecolog ical niche of the animal. One such function of hair is that it acts as a protection mechanism. Sea otters have specialized hairs that trap air close to the body to keep them dry and provide warmth, protecting them from t he cold temperatures of the water which they inhabit. A porcupine possesses modified hairs that ar e very rigid and barbed to provide defense against predators. A number of species possess hairs that are able to provide information about their surroundings. These modi fied hairs, or vibrissae, supply haptic feedback to the animal (Dykes, 1975). Vibri ssae are hair follicles that are surrounded by a blood filled sinus, bounded by a dense connective tissue capsule, robustly innervated, and provide somatosensory information (Dykes, 1975; Rice and Munger, 1986). 12

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Manatee Hair and Behavior Manatees possess a unique arrangement of specialized sensory hairs (vibrissae), present on the face and across the body, which is unique among mammals. All of the manatees hairs are tactile in nature. Manatees possess a very high density of vibrissae on their facial region, about 30 ti mes greater than the post-facial region. The lips of the manatee are prehensil e and they are able to evert the U2 and L1 FSCs (stout vibrissae located in the per ioral region) for use during feeding and to manipulate objects, termed oripulation (Marshall et al., 1998a; Reep et al., 1998). The number of axons per follicle decreases in locations dist al to the oral cavity (Reep et al., 2001). Vibrissae on the oral disk, classified as bristl e-like hairs that are in termediate in stiffness and innervation, are used to investigate objects and food items (Hartman, 1979; Marshall et al., 1998a). A previous study with the two Florida m anatees (the same subjects that were being utilized for this dissertation research) in vestigated the active touch ability of the manatees facial vibrissae. Weber fractions the percentage difference in size that is needed for the subject to detect a difference between objects, were generated and were compared to the Weber fractions of other sp ecies (Table 1-1). Th is measure of just noticeable difference allows for a comparison of sensitivity even though the methodology may be different. Both manatees demonstrated very low Weber fractions, one subject being able to detect differences in size down to 2.5% and the other subject down to 7.5%, which in conjunction with ot her sensory research demonstrates that manatees are tactile specialists (Bauer et al., 2012). 13

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Vibrissae Comparative Distribution and Innervation Vibrissae, commonly referred to as whiskers, are located primarily on the mystacial region of terrestrial and aquatic mammals. They can posses a number of mechanoreceptors such as Merkel cells, lanceolate endings, and free nerve endings (Zelena, 1994). A deep vibrissal nerve c ontaining 100 200 axons is found in rodents (Rice and Munger, 1986) whereas a number of aquatic mammals possess several main nerves, and an increased number of axons per follicle (Dehnhardt et al., 1999; Reep et al., 2001; Sarko et al., 2007a). Ringed s eals have between 1,000 and 1,500 axons per vibrissa (Hyvrinen, 1995) and bearded seals hav e a similar range, with a maximum of 1,650 (Marshall et al., 2006). The Australian wa ter rat, which lives on land but hunts for prey in water, displays an intermediate c ount of 500 axons per follicle, providing an interesting crossover between the two groupi ngs (Dehnhardt et al., 1999). The number of axons provides the opportunity for a great er somatosensory resolution; however the axonal branching beyond the FSCs is unknown. Although the manatee appears to have less axons per FSC in comparison to other s pecies, a summation of the full body results in ~210,000 axons (Reep et al., 1998), ecli psing other species that only possess mystacial vibrissae. Aquatic mammals have developed adaptatio ns to aid in obtaining information about their environment. Walruses use thei r stiff vibrissae to explore the benthic substrate in search of shellfish and are able to discriminate different objects at a fine scale (Fay, 1982; Kastelein and van Gaal en, 1988). Seals and sea lions have been found to discriminate fine differences in objects (Dehnhardt, 1994; Dehnhardt and Kaminski, 1995; Dehnhardt and Dcker, 1996). It has also been demonstrated that sea lions (Glser et al., 2011) and seals (Dehnhar dt et al., 2001; Schule-Pelkum et al., 14

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2007) can follow hydrodynamic trails generat ed by swimming objects such as prey or conspecific s. Manatees use their facial vi brissae to investigate food items and novel objects and uniquely possess post-facial vibrissae that may be used to detect hydrodynamic stimuli (Hartm an, 1979; Marshall et al., 1998; Bachteler and Dehnhardt, 1999; Reep et al., 2002). Vibrissae Comparative Function Aquatic mammals face a unique challenge that terrestrial mammals do not. The increased density of water in comparison with air causes a constant deflection of vibrissae during any movement. Hanke et al. (2010) noted that harbor seals possess vibrissae that have an undulated surface struct ure. This specialization results in reduced vibrissal vibration, and thus a reduc tion in self-noise during swimming. The efference copy mechanism, a method of negating self-generated movement at the sensory level to maintain tactile sensitivity to external stimuli, that fish employ could also be utilized by aquatic mammals to avoid sensory overload. As noted by Reep et al. (2002), pressure waves, either auditory or vibratory depending on frequency levels, travel almost five times fast er in water than in air (Urick, 1983). This would necessitate aquatic mammals being able to detect and proc ess stimuli much more rapidly, which could explain the increased number of axons per FSC seen in aquatic versus terrestrial species. Vibrissae in Manatees A Mammalian Lateral Line? Manatees inhabit the coastal and inland waters of Florida. Previous research has demonstrated good hearing (Ger stein et al., 1999; Mann et al., 2005) and noise localization (Colbert et al., 2009) abilities although their visual acuity is quite poor (Bauer et al., 2003). Manatees seem to have little difficulty navigating these turbid 15

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waterways which often contain obstacles which they must maneuver around (Hartman, 1979). Anatomical an d behavioral evidence suggests that the follicle sinus complexes (FSCs) that manatees possess on their ent ire body may form a sensory array system for detecting hydrodynamic stimuli, receivi ng cues from currents and in-water objects, analogous to the lateral line syst em of fish (Reep et al., 2002). This is consistent with anatomical and behavioral evidence that manatees are tactile specialists, evidenced by their specialized facial morphology and the use of these vibrissae during feeding and the active investigation/manipulation of objects. A mult i-phase behavioral research study has been initiated to gain a better understanding of how manatees utilize this unique tactile sensory system. Manatees have up to 250 axons per FSC of the facial region (Reep et al., 2001). The FSCs of manatees possess merkel endi ngs, a slowly adapting mechanoreceptor associated with low frequency vibration detecti on, that are found within the ring sinus and at the rete ridge collar in post-facial and bristle like hairs which may allow for multiple aspects of a stimulus and deflection intensities to be extracted (Rice et al., 1997; Ebara et al., 2002; Sarko et al., 2007a). Merkel cells in the post-facial FSCs were highly innervated in contrast to the faci al vibrissae (Sarko et al., 2007a) possibly highlighting a difference in use with the facial vibrissae as an active touch mechanism and the post-facial FSCs as a passive detection system. Sarko and colleagues (2007a) discovered a tangle nerve ending unique to manatees that might be a low threshold mechanoreceptor indicating a possibl e increased sensitivity of manatees to minute stimuli. Vibrissae on non-mystacial regions have been demonstrated to play a crucial role in some species. Naked mo le rats use modified hairs located on their 16

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bodies for orientation and some squirrels and jerboas possess tactile hairs on their extremities that could provide feedback about landing s ites after jumps (C rish et al., 2003; Sokolov and Kulikov, 1987). The post-facial portion of the manat ee body has approximately 3,000 FSCs dispersed across it (Reep et al., 2002). T hese vibrissae are distributed somewhat regularly and are hypothesized to provide feedback on hydrodynamic stimuli analogous to the lateral line system of a fish (R eep et al., 2002). This hypothesis has been supported both anatomically (Sarko et al., 2007a) and observationally. During low frequency trials of a behavioral audiogram, Gers tein et al. (1999) observed the test subject orienting its body in a manner so t hat the manatees body received the stimulus rather than the animals head. Similar positioning was seen during low frequency localization testing with the test subjects of this research, Hugh and Buffett (J. Gaspard, pers. obs.). Both manatees oriented t heir body towards the stimulus generating equipment rather than their fa cial vibrissae. These anecdotal remarks support Reep and colleagues (2002) in their not ion of an array of tactile re ceptors capable of detecting low frequency hydrodynamic stimuli which w ould provide a mechanism for manatees to gain information about their surroundings. In addition to detecting water movements such as those caused by rivers and spring heads as well as tidal flows, the total body coverage of sinus hairs might allow for the de tection of near field objects due to the flow fields that are generated as an animal moves pas t them (Hassan, 1989) The ability of any aquatic animal to navigate is crucial for it s survival and increases in complexity in a three-dimensional environment. The capacity to obtain this environmental information from a sensory system would provide a manatee with the needed feedback to perform 17

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simple tasks such as navigation and object a v oidance. The aims of this dissertation have been designed to explore the manatees passive tactile abilities to detect hydrodynamic stimuli, the role of the vibrissae (possibly singularly and as an array) within the sensory system, and their ability to determine directionalit y of hydrodynamic stimuli. Table 1-1. Weber Fractions of Asian el ephant (Dehnhardt et al., 1997), Antillean manatee (Bachteler and Dehnhardt, 1999), harbor seal (Dehnhardt et al., 1998), human (Morley et al., 1983), and the 2 manatees used in these studies, Hugh and Buffett (Bauer et al., 2012). Species Weber Fraction Asian elephant (trunk) 0.14 Antillean manatee (facial complex) 0.14 Harbor Seal (facial vibrissae) 0.09 Human (index finger) 0.04 Hugh (facial complex) 0.075 Buffett (facial complex) 0.025 18

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Project Objectives Chapter 2 Objective The objective to Chapter 2 was to determine the sensitivity of t he manatees facial vibrissae and their importance in t he detecti on of hydrodynamic stimuli Chapter 3 Objective The objective to Chapter 3 was to determi ne the sensitivity of the manatees postfacial vibrissae and their importance in the detection of hydrodynamic stimuli Chapter 4 Objective The objective to Chapter 4 was to determine the ability of manatees to discriminate the direction of hydrodynamic stimuli using post-facial vibrissae Significance of the Project This project creates a behavioral foundation to assess t he tactile abilities of manatees and the importance/role of the vibrissae, s upporting the hypothesis of manatees as tactile specialists. 19

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CHA PTER 2 DETECTION OF HYDRODYNAMIC STIMUL I BY THE FACIAL VIBRISSAE OF THE FLORIDA MANATEE ( TRICHECHUS MANATUS LATIROSTRIS ) Background Manatees possess a unique arrangement of specialized sensory hairs, classified as vibrissae, which are present on the face and across the body. Anatomical and neurophysiological evidence in conjunction with behavioral assessments from other species as well as manatees suggest that vi brissae play an important role in detecting environmental stimuli. Each vibrissal apparatus is known as a follicle-sinus complex (FSC), which includes a blood filled sinus, bounded by a dense connective tissue capsule, is robustly innervated, and provides haptic feedback to t he animal (Dykes, 1975; Rice et al., 1986). Vibrissae are located primar ily on the mystacial region of terrestrial and non-Sirenian aquatic mammals, and are commonly referred to as whiskers. They can posses a number of mechanoreceptors such as Merkel cells, lanceolate endings, and free nerve endings (Zelena, 1994). A deep vibrissal nerv e containing 100 200 axons is found in rodents (Rice et al., 1986), whereas a num ber of aquatic mammals possess several main nerves, and a higher number of axons per follicle (Dehnhardt et al., 1999; Reep et al., 2001; Sarko et al., 2007a). Ringed s eals have between 1,000 and 1,500 axons per vibrissa (Hyvrinen, 1995) and bearded seals ex hibit a similar range, with a maximum of 1,650 (Marshall et al., 2006). The Austra lian water rat, which lives on land but hunts for prey in water, displays a count of 500 axons per follicle, intermediate between terrestrial and aquatic species (Dehnhardt et al., 1999). Manatees have up to 250 axons per FSC of the facial regi on (Reep et al., 2001). 20

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The FSCs of manatees possess Merkel endings that are found within the ring sinus and at the rete ridge collar in post-facial and bristle like hairs which may allow for the extraction of multiple features of a stimulus, potentia lly including the intensity, direction, velocity, and accele ration of hair deflection (Rice et al., 1997; Ebara et al., 2002; Sarko et al., 2007a). Merk el cells in the post-facial FSCs are highly innervated in contrast to the facial vibri ssae (Sarko et al., 2007a), po ssibly implicating the facial vibrissae in active touch and the post-facial FSCs in a passive detection system. Sarko and colleagues (2007a) discovered a t angle nerve ending unique to manatees that might act as a low threshold mechanoreceptor, indicating a possible increase in sensitivity of manatees to mi nute stimuli. Vibrissae on non -mystacial regions have been demonstrated to play a crucial sensory role in some species. Naked mole rats use modified hairs located on their bodies for ori entation as they exist predominantly in burrows and possess poor vision (Crish et al ., 2003). Some squirrels and jerboas possess tactile hairs on their extremities that could provide feedback about landing sites after jumps (Sokolov and Kulikov, 1987). Aquatic mammals face a unique challenge th at terrestrial mamma ls do not. The increased density of water compared to air c auses a constant deflection of vibrissae during any movement. Marshall et al. (2006) noted that bearded seals possess vibrissae that are more rigid than in other s pecies and are oval in shape. The increased stiffness would allow for a reduction in vi brissal movement in an aqueous environment, and the unique contour of t he vibrissae would reduce hydrodynamic drag. The efference copy mechanism that has been do cumented in some fishes (Bell, 1982), allowing the organism to differentiate between externally generated stimuli versus those 21

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resulting from its own actions, could also be utilize d by aquatic mammals (Bell, 1982; Coombs et al., 2002). To aid in obtaining information about their environment, aquatic mammals have developed adaptations of vibrissa l systems. Walruses use their stiff vibrissae to explore the benthic substrate in search of shellfish and are able to discriminate different objects at a small scale (Fay, 1982; Kastelein and van Gaalen, 1988). Seals and sea lions have been found to discriminate fine differences in objects and accurately track the hydrodynamic trails generated by prey (D ehnhardt, 1994; Dehnhardt and Kaminski, 1995; Dehnhardt and Dcker, 1996; Dehnhardt et al., 1998; Dehnhardt et al., 2001; Schule-Pelkum et al., 2007). Manatees use t heir facial vibrissae to investigate food items and novel objects (Hartman, 1979; Marshall et al., 1998; Bachteler and Dehnhardt, 1999; Reep et al., 2002). They may also use them to detect hydrodynamic stimuli. Manatees possess vibrissae across their entire body, which is unique among mammals, though hyraxes appear to have a similar arrangement (D. Sarko, pers. comm.). Vibrissae are ~30 times denser on the facial r egion than on the post-facial body. The lips of the manatee are very mobile and prehensile. The stout vibrissae on the upper lip (U2 field) and lower lip (L1 fiel d) are everted during grasping of objects, including plants ingested during feeding. This oral grasping has been termed oripulation (Marshall et al., 1998; Reep et al., 1998). Th e number of axons per follicle decreases when traveling further from the oral cavity (Reep et al., 2001). Vibrissae on the oral disk, classified as bristle-like hairs that ar e intermediate in stiff ness and innervation, are 22

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used in non-grasping investi gation of objects and food it ems (Hartman, 1979; Marshall et al., 1998). A previous study with the same two Flori da manatees used in the current research investigate d their ability to perfo rm active touch discrimination using the facial vibrissae. Weber fractions (just-noticeable-differences), the percentage chan ge in size needed for the subject to detect a difference between objects, were measured and compared to those of other species. Both manatees dem onstrated very low Weber fractions. One subject was able to detect differences in si ze down to 2.5% and the other subject down to 7.5% (Bauer et al., 2012). The present study sought to test the hypothesis that manatees use their facial vibrissae not only for active touch but also to detect hydrodynamic stimuli. We c onducted three experiments to te st this hypothesis. The first generated a manatee tactogram, tactile detection thresholds across a set of low frequencies. A second test re stricted vibrissae to assess their involvement in detection of hydrodynamic stimuli. A third experiment assessed vibrissae sensitivity using a signal detection format. Materials and Methods Subjects The subjects were two male Florida manatees ( Trichechus manatus latirostris ) housed at Mote Marine Laboratory & Aquarium in Sarasota, Florida, USA. Buffett and Hugh, 21 and 24 years of age, respectively, at the initiation of the study, had an extensive training history in the context of husbandry and sensory research (Colbert et al., 2001; Bauer et al., 2003; Mann et al., 2005; Colbert et al., 2009; Bauer et al., 2012; Gaspard et al., 2012). 23

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Experiment I Tacto gram The tactogram established the tactile thres holds for frequencies ranging from 5 Hz 150 Hz. The upper limit was selected to mini mize the possibility that detection of the stimuli by hearing confounded ta ctile measurements. Procedures The manatees were trained utilizing operant conditioning through positive reinforcement to signal the detection of hydr odynamic stimuli direct ed at their facial vibrissae. A go/no-go procedure was used to determine stimulus detection. If the stimulus was detected, the manatee re sponded by withdrawing from the horizontal stationing bar and touching a response paddle 1 m to the subject s left (go response), lateral to the head, with its muzzle. If no st imulus was detected, the manatee remained at station for a minimum of 10 seconds no-go response (Figure 2-1). Correct responses were followed by an auditory secondary reinforcer, a digitized whistle from an underwater speaker, followed by primary reinforcement, preferred food items of pieces of apples, carrots, beets, and monkey biscuits. After a correct response on a signal present trial, the int ensity of the stimulus was a ttenuated 3 dB. If the manatee was incorrect on a signal present trial, the in tensity level of the stimulus was increased by 3 dB. A staircase method (Cornswee t, 1962), noted by a decrease in stimulus intensity following a correct response for a pr esentation trial or an increase in stimulus intensity following an incorrect choice on a presentation trial, was used in which eight reversals determined a threshold measurement. Four warm-up trials were conducted prior to testing to assess the motivation and performance levels of the manatees with the stimulus at the same fr equency and highest level that was to be tested. A criterion of 75% correct on warm-up trials had to be me t in order for testing to occur during that 24

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particular session. If the subjec t failed to meet criterion on t he first set of warm-up trials, a second warm-up set was conducted. Testi ng was not conducted if the subjec t failed to meet criterion on the second warm-up block. The subjects were trained to station by placing their postnasal crease on a horizontal PVC bar (2.5 cm diameter) at a depth of 0.75 m, 10 cm both forward and below, on the midline, from t he stimulus generating sphere (Figure 2-2). A tri-cluster LED signaled the initiation of every trial, illu minating for a duration of 1 s, followed by a 0.5 s delay prior to both signal present and signal absent windows. The stimuli were generated by a 5.7 cm sinusoidally oscillating sphere driven by a computer-controlled calibrated vibration shaker. The sphere was connected to the shaker via a rigid stainless steel rod. The shaker and attachment rod were oriented vertically in the water column. The stimuli were 3 seconds in duration with cos2 rise-fall times of 300 ms and ranged from 5 150 Hz. Signal present versus signal absent trials were counterbalanced using a 1:1 ratio. Daily sessions (weekdays) were conducted with each session focused on a single frequency, encompassing 12 72 trials. A single frequency was tested over the course of 2 separate staircase sessions conducted on consecutive days to confirm thresholds. If the thresholds were not within 6 dB of each other, a third session was condu cted and the thresholds were averaged. An underwater speaker presented masking noise throughout the session to mask any auditory artifacts generated by the shaker. The speaker also presented the secondary reinforcer when the manatee was correct on a trial. Equipment A dipole vibration shaker (Data Physics Signal Force, Model V4, San Jose, CA, USA) with a 5.7 cm diamet er rubberized sphere connect ed via a 35.6 cm, rigid, 25

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stainless steel extension rod was used to generate the stimuli. The dipole shaker generates a localized flow that dec reases in amplitude as 1/distance3, as opposed to a monopole source that decreases in amplitude as 1/distance2 (Kalmijn, 1988). To eliminate any vibrational transfer between the shaker apparatus and the stationing apparatus, the stationing apparat us and the shaker mount were separate pieces of equipment buffered with shock absor bing foam (Figure 2-3). The stimuli were generated digitally by a Tucker-Davis Technologies (TDT) Enhanced Real-Time Processor (RP2.1, Alachua, FL, USA; sample rate 24.4 kHz), attenuated with a TDT Programmabl e Attenuator (PA5) to c ontrol level, and amplified with a Samson Power Amplifier (Servo 120a, Hauppauge, NY, USA). The signal generating equipment was controll ed by a program in MATLAB (MathWorks, Natick, MA, USA) in conjunction with a graphical us er interface (TDT Real-Time Processor Visual Design Studio) created specifically for this research. A digital output on an RP2.1 was used to control the LED that indicated the start of a trial. A separate D/A channel was used to generate the acoustic secondary reinforcer, which was presented through an underwater speaker (Clark Synthesis, Model AQ-39, Littleton, CO, USA) when the manatee was correct on a trial. The speaker was located >1 m away from the subject and also presented noise (151 dB re 1 Pa; 12.2 kHz bandwidth) constantly through the session to mask any auditory artifacts from t he generation of the hydrodynamic stimulus. These signals were amplified by a separate amplifier (American Audio, Model VLP 300, Los A ngeles, CA, USA) to avoid crosstalk. For stimuli analysis and calibration, a 3-dimensional accelerometer (Dimension Engineering, Model DE-ACCM3D Akron, OH, USA) was em bedded into the sphere to 26

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measure its movement. MATLAB was used to calculate, plot, and log the stimulus for each trial. This accelerometer was used to monitor the shaker operat ion during testing. To calculate particle motion from the di pole for threshold measurements, a 3-D accelerometer was mounted to a neutrally buoyant, spring-mounted geophone. The outputs from all three channels were recor ded simultaneously by the RP2.1. The rms acceleration of the unattenuated stimulus for each stimulus frequency was calculated from these recordings. The magnitude of acceleration from all three axes was calculated as the square root of the sum of squares of each axis. The acceleration at the threshold was calculated by scaling the acceleration measured at no attenuation by the attenuation at threshold. For sinusoidal signals, particl e velocity is the particle acceleration divided by 2 f, and particle displacement is particle velocity divided by 2 f. The sensitivity of the accelerometer was veri fied by comparing its output when directly vibrated with the output of a laser vibrometer pointed at the accelerometer (Polytec, CLV 1000, Irvine, CA, USA). The laser vi brometer could not be used in the manatee tank because it only measures motion in one direction along the laser beam. To ensure that the test subjects we re not cued during testing, a number of protocols and measurements were conducted. A 3-D accelerometer was routinely attached to the stationing apparatus to ensur e that there was no vi brational transfer from the shaker during pres entation trials. The posit ion of the manatee on the stationing apparatus prevented any direct visual cues from the oscillating sphere. Furthermore, the subjects minimum angle of resolution (Bauer et al., 2003) was greater than the movement of the sphere, which subtended 20 arc minutes for Buffett and 22 arc minutes for Hugh. The difference was attributable to the greater distance of 27

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Buffetts eyes than Hughs from the front of his face. R esearchers were unable to detect the movement of the sphere visually at the manatees thresholds for most frequencies. The research trainer responsible for verifying the position of the manatee and providing the primary reinforcement was blind to whether the ensuing trial was a stimulus-present or stimulus-absent trial. This trainer was also out of the manatees direct line of sight and remained motionle ss until the trial sequence was complete. Experiment II Restriction Tests Experiment 1 established the detection thre sholds for hydrodynamic stimuli. The function of vibrissae in det ection was not assessed. To determine if the vibrissae contributed to detection of the hydrodynamic stimuli, tests in which vibrissae were restricted with variable size mesh netting were conducted using the same procedure as in Experiment I. The mesh was arranged on a stainless steel ring slightly larger than the manatees muzzles and mounted on the stat ioning apparatus. The subjects were trained to insert their muzzle into the mesh, which restricted a percentage of their facial vibrissae exposed to particle flow (Figure 2-4). The threshold testing was conducted under four masking conditions, ~10%, ~25%, ~65%, and ~100% of vibrissae restricted determined by the sizes of the openings in the mesh (Table 2-1). The number of vibriss ae protruding through the mesh were counted during 3 separate placements and verified by a second counter for each mesh condition to determine the percentage occluded. Experiment III Signal Detection Threshold measures are influenced by decis ion criteria. An alternative way to address sensitivity while controlling for these cr iteria is to use a signal detection analysis (Gescheider, 1997). Detection testing was conducted under two conditions, with and 28

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without the fine mesh (0.397 mm), at 25 Hz at 0.21 m di splacement, a 3.35x (10.5 dB) attenuation from the starting level during thres hold testing. Fifty trials were conducted under each condition (25 signal present; 25 signal absent). Values for d' and C were calculated. In signal detection theory d' is an unbiased sensitivity parameter. C is an index of the decision criterion. Unbiased responses are indicated by C values approaching zero. Values of C less than 0 indicate a greater probability of reporting a signal present when it is not, a false alarm, and values greater than 0 indicate a greater probability of reporting a signal absent when it is in fact present (Gescheider, 1997). Results Experiment I Tactogram Results for the behavioral tactogram hi ghlight the sensitivity and frequency dependence of the detection of hydrodynamic st imuli (Table 2-2). Threshold values were calculated in terms of displacement, ve locity, and acceleration as it is unknown which stimulus(i) the manatees detect. Both subjects displayed thresholds below 1 micron of particle displacement for frequencies above 10 Hz. At 150 Hz Buffett and Hugh detected particle displacement near and below 1 nm, respectively, using their facial vibrissae. Sensitivity was positively correlated with frequency with a decrease in sensitivity for stimuli at 10 Hz and below, high lighted by the failure to detect the stimulus at 5 Hz by one subject (Figure 2-5, 2-6, 2-7). Both manatees demonstrated similar thresholds, suggesting that the co mbined tactogram may be a reasonable representation of the abilities of manat ees generally. Over 20 sessions were videotaped underwater to view the side profil e of the manatees and showed that they did not appear to flare their mu zzle to expose their perioral vibrissae during testing, with the BLHs composing the dominant class of fa cial vibrissae exposed to the stimuli. 29

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Experimen t II Restriction Tests Data from the restriction trials demonstr ated that the thresholds increased as a greater number of vibrissae were restrict ed (smaller mesh hole size) (Table 2-3). Because of the low sample size (n=2 manatees) only descriptive statistics have been calculated. The regression coefficients a ll have positive slopes and most show high coefficients of determination (all but one have r2>0.6) when the fraction of vibrissae restricted is regressed against the displacement threshold (Table 2-3). However, at the higher frequencies, the thresholds did not show as much of an effe ct of restriction Figures 2-8, 2-9). Interesti ngly, the manatees were unable to detect the stimuli at lower frequencies as a greater perc entage of the vibrissae were restricted, as Buffett demonstrated no response to the stimuli at 25 Hz (1.69 m displacement) and Hugh could not detect the stimuli at 25 or 50 Hz (0.44 m displacement) when the 35 micron mesh was employed. Experiment III Signal Detection A signal detection analysis was conducted wit h trials run at the same frequency (25 Hz) and level (0.21 m displacement) highlighting the re striction of vibrissae as the only difference between tests. The d' and C values were calculated for both the no mesh and fine mesh conditions (Table 2-4). The value of d' decreased from 1.80 to 0.91 when the fine mesh was added into the pr ocedure, restricting > 65% of the facial vibrissae. This indicated that the mesh was reducing the sensitivity, therefore suggesting the importance of t he vibrissae in detecting hy drodynamic stimuli. The positive C values for both c onditions demonstrate that th e manatees decisions were conservative and probably provide an underes timation of their tactile abilities. 30

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Discussio n The thresholds determined for the faci al vibrissae of manatees demonstrate remarkable sensitivity, highlighted by the det ection of particle displacement approaching and below 1 nanometer at 150 Hz. Dehnhardt and colleagues (1998), in a study which served as a model for this one, tested the ability of a harbor seal ( Phoca vitulina ) to detect hydrodynamic stimuli. Our results i ndicate that manatees are more sensitive than harbor seals by an order of magnitude (Figure 2-5) and more recent research has established that the California sea lion ( Zalophus californianus ) has an intermediate sensitivity (Dehnhardt and Mau ck, 2008). Comparing the th resholds for these three species as a function of displacement, veloci ty, or acceleration reveals a much larger range for displacement than for velocity or a cceleration. We do not know which of these parameters are sensed by the vi brissae. Studies with rat vi brissae suggest that they are velocity-sensitive because thresholds vari ed as a function of stimulus amplitude or frequency, but not as a f unction of amplitude*frequency (Adibi et al., 2012). As a greater percentage of the vibrissae were limited, the manatees thresholds increased and the subjects were not able to detect the stimuli at the lower frequencies when they were completely restricted. T hese results strongly suggest that tactile senses, including those mediated by the vibr issae, were responsible for the observed thresholds, and not some other sense such as vision or hearing. MARs for both animals (Bauer et al., 2003) were above the angle of resolution necessary to see the distance moved by the stimulus sphere displa cement. Auditory thresholds of manatees are highest at low frequencies (Gerstein et al., 1999; Gaspard et al., 2012). Note that one of the two manatees tested by Gerstein (1999) could detect the acoustic signals from 15 400 Hz with thresholds from 93-111 dB re 1Pa. However, Gerstein et al. 31

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(1999) suggested that under 400 Hz the manat ee was detecting the stimulus tactually, rather than by hearing, based on re sponse characteristics. In the restriction experiments, there was convergence of sensitivity at the higher frequencies. The mechanism of detection may change at these frequencies, and could involve follicle-associated mechanoreceptors and surface Merkel cells. The increase of thresholds during restriction testing and the dec rease in d' with the inclusion of the mesh netting during signal detection tests indicates that the vibrissae were a key component of the detection of low frequency vibratory stimuli. It is not known what cues manatees use fo r orientation as they navigate through their environment and migrate between summer and winter refugia. They spend a significant portion of time in turbid waters especially during travel, but they have poor visual acuity (Mass et al., 1997, 2012; Bauer et al., 2003) and do not echolocate. Previous work has shown that the perioral br istles play a dominant role during feeding and oripulation (Hartman, 1979; Marshall et al., 1998; Bachteler and Dehnhardt, 1999; Bauer et al., 2012). The bristle like hairs of the oral disk (BLH) may serve as a sensory array to detect hydrodynamic stimuli, in addit ion to their use in direct contact tactile scanning (Bauer et al., 2012). The anatomical differentiation between the stout perioral bristles and the more pliant BLHs supports the likelihood of a ro le for the latter in passive detection of hydrodynamic stimuli (Sarko et al., 2007a) as does the test subjects posture during testi ng. In the present study t he manatees did not attempt to flare their lips to present the perioral vibrissa e, thus the stimuli we re directed primarily toward the BLHs. 32

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Bearded seals and ringed seals possess FS Cs innervated by more than 1,000 axons per vibrissa (Hyvrinen, 1995; Mars hall et al., 2006) with rodents demonstrating significantly less innervation at 100 200 per FSC (Rice et al., 1986). The Australian water rat, since it does not live exclusiv ely in an aquatic environment, and displays an intermediate number of axons per follicle (~500) seems to optimize its existence in both media (Dehnhardt et al., 1999). The increased innervation of aquatic species highlights the specialization required to exist in a comp lex environment. The facial region of the manatee is densely populated with approxim ately 2,000 vibrissae, collectively innervated by over 100,000 axons. Approximately 600 of these facial vibrissae are the BLHs located on the oral disk (Reep et al., 1 998; 2001). This axonal innervation, up to 250 axons per facial vibrissae, is comparable to the specialized nasal region of the star nosed mole (Catania and Kaas, 1997). Sarko and colleagues (2007a) f ound that the dense distribut ion of Merkel endings may provide a specialized mechanism for dete cting the directiona l deflection of the follicle. Novel receptors discovered in manatee FSCs may also be an adaptation for detecting stimulus features in an aquatic environment, including minute perturbations and directionality. These peripheral specializations of the manatee somatosensory system are supported by larger regions of t he somatosensory brainstem, thalamus, and cerebral cortex. Several cortical regi ons exhibit specialized neuronal aggregations (Rindenkerne) which appear to be analogous to the barrel cortex associated with vibrissae representations in rodents (R eep et al., 1989; Marshall and Reep, 1995). Behavioral studies with mottled sculpin ( Cottus bairdi ) using a dipole stimulus found acceleration thresholds of about 0.18 mm/s2 for 10-100 Hz (Coombs and 33

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Janssen, 1989a; 1989b; 1990). This is about 4-20 times more sensitive than the manatee facial vibriss ae thresholds over the same frequency range. Several studies have investigated the ability of fish to detect particle displacement; however these responses were primarily measured in pr imary auditory afferents, and were thus associated with perception of ac oustic stimuli. Oscars ( Astronotus ocellatus ) detected particle displacement of 1.2 1.6 nm (RMS) at 100 Hz (Lu et al., 1996). Similar sensitivity was demonst rated by goldfish ( Carassius auratus ) and toadfish ( Opsanus tau ) with a detection of particle displacement less than 1 nm (RMS) (Fay and Olsho, 1979; Fay, 1984; Fay et al., 1994). Particle di splacement sensitivit y for the manatees at 100 Hz was 1.9 nm and 3.1 nm (Table 2-2). Although the detection modality sometimes differed in fish, the manatees were slightly less sensitive in the detection of particle displacement. Blind cavefish sense objects in the wate r by detecting alterations in self-produced hydrodynamic stimuli as they near or pass them (Campenhausen et al., 1981; Weissert and Campenhausen, 1981; Hassan, 1989). Future research should investigate whether manatees utilize their own self-generated hydr odynamic stimuli in a similar manner to the blind cave fish, detecting reflected bow waves or interruptions of the pressure waves, gaining information about their typically turbid environment. 34

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Table 2-1. Hole siz e of mesh netting and th e approximate percentage of facial vibrissae that were restricted. Mesh Hole Size Percentage of Vibrissae Restricted Large (3.175 mm) ~10% Intermediate (1.588 mm) ~25% Fine (0.397 mm) ~65% 35 microns (0.035 mm) ~100% 35

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36 Table 2-2. Facial threshold values and false alarm rate for each tested frequency for Buffett and Hugh. Note that Hugh was not able to detect the stimuli at 5 Hz. Buffett Frequency (Hz) Displacement ( m) Velocity (mm/s) Acceleration (mm/s2) False Alarm Rate 5 4.2162 0.1325 4.1613 0.13 10 1.0786 0.0678 4.2582 0.11 15 0.3095 0.0292 2.7493 0.13 20 0.1741 0.0219 2.7493 0.05 25 0.1503 0.0236 3.7087 0.10 50 0.0385 0.0121 3.7951 0.04 75 0.0079 0.0037 1.7548 0.00 100 0.0019 0.0012 0.7400 0.14 125 0.0031 0.0024 1.9021 0.04 150 0.0013 0.0012 1.1728 0.00 Table 2-2. Continued. Hugh Frequency (Hz) Displacement ( m) Velocity (mm/s) Acceleration (mm/s2) False Alarm Rate 10 1.5236 0.0957 6.0148 0.00 15 0.3095 0.0292 2.7493 0.00 20 0.1465 0.0184 2.3133 0.04 25 0.1503 0.0236 3.7087 0.00 50 0.0343 0.0108 3.3824 0.02 75 0.0040 0.0019 0.8795 0.03 100 0.0031 0.0020 1.2423 0.06 125 0.0026 0.0020 1.6004 0.04 150 0.0009 0.0009 0.8303 0.09

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Table 2-3. Displacem ent thresholds ( m) for each frequency (Hz) based on mesh size. An asterisk (*) designates that the subj ect did not respond to the presentation of the stimulus under the conditions. The false alarm rates for each frequency and condition are presented in parentheses. Coefficient of determination values (r2) for the regression of t he fraction of vibrissae restricted versus displacement th reshold were also calculated. Buffett Frequency No Mesh Large Mesh Intermediate Mesh Fine Mesh 35 Micron Mesh r2 25 0.1503 (0.10) 0.1865 (0.03) 0.3564 (0.07) 0.4822 (0.08) 0.93 50 0.0385 (0.04) 0.0385 (0.00) 0.1531 (0.11) 0.0684 (0.09) 0.7243 (0.06) 0.67 75 0.0079 (0.00) 0.0056 (0.05) 0.0158 (0.21) 0.0125 (0.07) 0.0223 (0.04) 0.71 100 0.0019 (0.14) 0.0053 (0.03) 0.0075 (0.09) 0.0053 (0.14) 0.0125 (0.01) 0.68 Table 2-3. Continued. Hugh Frequency No Mesh Large Mesh Intermediate Mesh Fine Mesh 35 Micron Mesh r2 25 0.1503 (0.00) 0.2123 (0.04) 0.5257 (0.00) 0.7112 (0.04) 0.90 50 0.0343 (0.02) 0.0543 (0.04) 0.1288 (0.06) 0.0912 (0.00) 0.30 75 0.0040 (0.03) 0.0047 (0.03) 0.0112 (0.00) 0.0133 (0.02) 0.0236 (0.00) 0.93 100 0.0032 (0.06) 0.0032 (0.09) 0.0063 (0.00) 0.0044 (0.09) 0.0193 (0.03) 0.70 37

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Table 2-4. Signal Det ection Theory anal ysis of testing under no mesh and fine mesh conditions for Buffett. Trials we re conducted at 25 Hz at 0.21 m displacement. Fifty tria ls were conducted under each condition (25 signal present; 25 signal absent) No Mesh Yes No d' C Signal Present 0.52 0.48 Signal Absent 0.04 0.96 1.80 0.85 Table 2-4. Continued. Fine Mesh Yes No d' C Signal Present 0.20 0.80 Signal Absent 0.04 0.96 0.91 1.30 38

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Figure 2-1. Correct response to a signal pr esent trial (on left), with the manatee leaving station and depressing the response paddl e, and a signal absent trial (on right), with the manatee rema ining stationed, during training trials. (Photos courtesy of author) 39

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Figure 2-2. Manatee stati oned with postnasal crease on horizontal white PVC bar orienting towards the stimuli generating sphere during tr aining trials. Note the response paddle in the foreground. (Photos courtesy of author) 40

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Figure 2-3. Experimental se tup showing the black PVC stationing apparatus, the vibration shaker housed in the separate aluminum frame, and the response paddle. (Photos courtesy of author) 41

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42 Figure 2-4. Testing set-up showing the m anatee stationed with its muzzle in a mesh netting, restricting a perc entage of the facial vibrissae. (Photos courtesy of author)

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0.0001 0.001 0.01 0.1 1 10 100 1101001000Frequency (Hz)Displacement ( m) Buffett Hugh Harbor seal Figure 2-5. Threshold values for displacement detection for bot h manatee test subjects Buffett (solid diamond, solid line) and Hugh (open circle, dashed line). Threshold values for a harbor seal (X) have been included for comparison (Dehnhardt et al., 1998). Both th e x-axis and y-axis are repr esented with logarithmic scales. 43

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0.0001 0.001 0.01 0.1 1 10 1101001000Frequency (Hz)Velocity (mm/s) Buffett Hugh Harbor seal Figure 2-6. Threshold values for velocity detection for both manatee test subjects Buffett (solid diamond, solid line) and Hugh (open circle, dashed line). Threshold values for a harbor seal (X) have been included for comparison (Dehnhardt et al., 1998). Both th e x-axis and y-axis are repr esented with logarithmic scales. 44

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45 Figure 2-7. Threshold values for acceleration detection for bot h manatee test subjects Buffett (solid diamond, solid line) and Hugh (open circle, dashed line). Threshold values for a harbor seal (X) have been included for comparison (Dehnhardt et al., 1998). Both the x-axis and y-axis are r epresented with logarithmic scales. 0.1 1 10 100 1000 1101001000Frequency (Hz)Acceleration (mm/s2) Buffett Hugh Harbor seal

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0.001 0.01 0.1 1 0255075100 No Mesh Large Mesh Intermediate Mesh Fine Mesh Displacement ( m)Fre quency (Hz)35 Micron Mesh was scaled logarithmically. Figure 2-8. Plot of displacement versus frequency for the 5 mesh conditions for Buffe tt. The y-axis 46

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0.001 0.01 0.1 1 0255075100 Frequency (Hz)Displacement ( m) No Mesh Large Mesh Intermediate Mesh Fine Mesh 35 Micron Mesh Figure 2-9. Plot of displacement versus frequency for the 5 mesh conditions for Hu gh. The y-axis was scaled logarithmically. 47

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CHA PTER 3 DETECTION OF HYDRODYNAMIC STIMUL I BY THE POST-FACIAL VIBRISSAE OF THE FLORIDA MANATEE ( TRICHECHUS MANA TUS LATIROSTRIS ) Background Manatees possess a unique arrangement of s pecialized sensory hairs, classified as vibrissae, present on the face and across the body. They possess a number of mechanoreceptors such as Merkel cells, lanceolate endings, and free nerve endin (Zelena, 1994). Vibrissae on non-mystacial regions have been demonstrated to play a crucial role in some species. Naked mo le rats use modified hairs located on their bodies for orientation as they primarily exist in burrows wher e cues other than tactile are limited (Crish et al., 2003) Aquatic mammals face a unique challenge th at terrestrial mammals do not. The increased density of water in comparison with air causes a constant deflection of vibrissae during any movement. Marsha ll et al. (2006) noted that bearded seals possess vibrissae that are more rigid than in other species and are oval in shape. The increased stiffness would allow for a reducti on in vibrissal mo vement and the uniqu contour of the vibrissae would reduce hy drodynamic drag, providing a method to compensate for aquatic life. The efference c opy mechanism that fish employ, allowing the organism to differentiate between externally generated stim uli versus those resulting from its own actions, could also be utilized by aquatic mammals (Bell, 1982; Coombs et al., 2002). To aid in obtaining information about their environment, aquatic mammals ha developed adaptations of vibrissa l systems. Walruses use their stiff vibrissae to explore the benthic substrate in search of shellfish and are able to discriminate different objects at a small scale (Fay, 1982; Kastelein and van Gaalen, 1988). Seals and sea lions gs e ve 48

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have been found to discriminate fine d bjects and accurately track the hydrodyn ki, 1995; Dehnhardt and Dcker, 1996; Dehnhardt et al., 1998; Dehnhardt et al., 2001; Schu ic 0 to 40 mm apart, about the same as the length of the hair. Mana e ~30 r nd f the locations was compared. Two underwater red lasers (Lasermate, Mode a ifferences in o amic trails generated by prey (D ehnhardt, 1994; Dehnhardt and Kamins le-Pelkum et al., 2007). Manatees use t heir facial vibrissae to investigate food items and novel objec ts (Hartman, 1979; Marshall et al., 1998; Bachteler and Dehnhardt, 1999; Reep et al., 2002). They may also use them to detect hydrodynam stimuli. Manatees have over 3,000 vibrissae across their post-facial body which are innervated by over 100,000 axons (Reep et al., 2001). The vibrissae are somewhat regularly distributed about 2 tees and their close evolutionary relative, the rock hyrax, are the only species known to have sinus hairs all over the body (D Sarko, pers. comm.). Vibrissae ar times denser on the facial region than on the post-facial body. Two Florida manatees were trained to detect hydrodynamic stimuli directed at thei post-facial body. The procedural design is si milar to that reported in Chapter 2 with several exceptions. The vibration shaker was enclosed in a waterproof housing a was located, primarily, on t he right side of the manatee, oriented horizontally in the water column and directed at the mid-body, dorso -laterally. A range of frequencies, 5 150 Hz, were tested to determine thresholds for several specific sites on the body. The sensitivity o l SL6505M) were attached to the shaker mount to allow for t he measurement of set distance of the manatee from the sphere, 20 cm. A waterproof camera (HelmetCamera, Sony 560 line cam) was mounted to the top of the shaker mount frame 49

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to record the manatees movements and distanc e of the test site on the manatee fro the sphere. Materials and Methods Subjects The subjects were two male Florida manatees ( Trichechus manatus lati housed at Mote Marine Laboratory & Aquarium in Sarasota, Florida, USA. Buffett and Hugh, 23 and 26 yea m rostris ) rs of age respectively at the initiation of the study, have an exten ert et 2; y that detection of the stimu as ined at secondary reinforcer, a digitized whistl e from an underwater speaker, followed by sive training history in the context of husbandry and research behaviors (Colb al., 2001; Bauer et al., 2003; Mann et al., 2005; Colbert et al., 2009; Bauer et al., 201 Gaspard et al., 2012). Experiment I Tactogram The tactogram established the tactile thres holds for frequencies ranging from 5 Hz 150 Hz. The upper limit was selected to mini mize the possibilit li by hearing confounded ta ctile measurements. Procedures The manatees were trained utilizing operant conditioning through positive reinforcement to signal the detection of hydr odynamic stimuli direct ed at their facial vibrissae. A go/no-go procedure was used to determine stimulus detection. If the stimulus was detected, the manatee re sponded by withdrawing from the horizontal stationing bar and touching a response paddle on the same side that the stimulus w presented with its muzzle. The response paddl es were located 1 m lateral to the head on either side of the subject. If no stimulus was detected, the manatee rema station for a minimum of 10 se conds. Correct responses we re followed by an auditory 50

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primary rei nforcement, prefer red food items of pieces of apples, carrots, beets, and monkey biscuits. After a correct response on a signal present trial, the intensity of the stimulus was attenuated 3 dB. If the manatee was incorrect on a signal present trial, y level of the stimulus was increased by 3 dB. A staircase method (Corn iter ion on the first set of warm-up trials, a second warm-up set not conducted if t he subject failed to m eet criterion on the seco m, 10 cm both forward and midline, from t he stimulus generating sphere (Figure 3-1). A tri-cluster LED by a ed h l the intensit sweet, 1962) was used in which ei ght reversals determined a threshold measurement. Four warm-up trials were conducted prior to testing to assess the motivation and performance levels of the manatees with the stimulus at the same frequency and highest level that was to be test ed. A criterion of 75% correct on warmup trials had to be met in order for testing to occur during that particular session. If the subject failed to meet cr was conducted. Testing was nd warm-up block. The subjects were trained to station by placing their postnasal crease on a horizontal PVC bar (2.5 cm diameter) at a depth of 0.75 below, on the signaled the initiation of every trial, illu minating for a duration of 1 s, followed 0.5 s delay prior to both signal present and signal absent windows. The stimuli were generated by a 5.7 cm sinusoidally oscillating sphere driven by a computer-controll calibrated vibration shaker. The sphere was connected to the shaker via a rigid stainless steel rod. The shaker and attach ment rod were oriented horizontally in the water column. The shaker was housed in a water-tight cylindrical housing with the rod passing through a sealed silicone barrier. T he stimuli were 3 seconds in duration wit cos2 rise-fall times of 300 ms and ranged from 5 150 Hz. Signal present versus signa 51

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absent trials were counterbalanc ed using a 1:1 ratio. Daily sessions (weekdays) wer conducted with each session focused on a single frequency, encompassing 12 72 trials. A single frequency was tested over t he course of 2 separate staircase sessions conducted on consecutive days to confirm thres holds. If the thresholds were n a factor of two (i.e. 6 dB) of each other, a third session was conducted and the thresholds were averaged. An underwater speaker presented masking noise throughout the session to mask any auditory artifacts generated by the shaker. Th speaker also presented the secondary rein forcer, or bridge, when the manatee was correct on a trial. Four locations on the manatees post-facial body were tested, all dorso-ventrally centered: three on the right side (forward th ird, middle third, and rear third) and the forward third of the left s e ot within e ide (Figure 3-2) To ensure that t he same region of the mana nd in A, foam. tee was tested on different days, t he position of the equipment was marked a repeated during each regional tes t. This was done for each mana tee as they differed size. Equipment A dipole vibration shaker (Data Physics Signal Force, Model V4, San Jose, C USA) with a 5.7 cm diameter rubberized sphere connected via a rigid stainless steel extension rod was used to generate the st imuli. The dipole shaker generates a localized flow that decreases in amplitude as 1/distance3, as opposed to a monopole source that decreases in amplitude as 1/distance2 (Kalmijn, 1988). To eliminate any vibrational transfer between the shaker and t he manatee, the stationing apparatus and the shaker mount were separate pieces of equipment buffered with shock absorbing 52

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The stimuli were generated digitally by a Tucker-Davis Technologies (TDT) Enhanced Real-Time Processor (RP2.1, Alachua, FL, USA; sample rate 24.4 kHz), attenuated with a TDT Programmabl e Attenuator (PA5) to c ontrol level, and amplified with a Samson Power Amplifier (Servo 120a, Hauppauge, NY, USA). The signal generating equipment was controll ed by a program in MATLAB (MathWorks, MA, USA) in conjunction with a graphical us er interface (TDT Real-Time Proce Visual Design Studio) created specifically for this research. A digital output on an RP2.1 was used to control the LED that indicated the start of a trial. A separate D/A channel was used Natick, ssor to generate the acoustic secondary reinforcer, which was presented throu the tly can Audio, Model VLP 300, Los A ngeles, CA, USA) to avoid crosstalk. uli analysis and calibration, a 3-dimensional accelerometer (Dimension Engin r esting. gh an underwater speaker (Clark Synthesis, Model AQ-39, Littleton, CO, USA) when the manatee was correct on a trial. The speaker was located >1 m away from subject and also presented noise (151 dB re 1 Pa; 12.2 kHz bandwidth) constan through the session to mask any auditory artifacts from t he generation of the hydrodynamic stimulus. These signals were amplified by a separate amplifier (Ameri For stim eering, Model DE-ACCM3D Akron, OH, USA) was em bedded into the sphere to measure its movement. MATLAB was used to calculate, plot, and log the stimulus fo each trial. This accelerometer was used to monitor the shaker operat ion during t To calculate particle motion from the di pole for threshold measurements during the initial post-facial sensitivit y testing, a 3-D accelerometer was mounted to a neutrally buoyant, spring-mounted geophone. The outputs from all three channels were recorded simultaneously by the RP2.1. The rms a cceleration of the unattenuated stimulus for 53

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each res so idal r e ise were of hydro y e stimulus frequency was calculated from these recordings. The magnitude of acceleration from all three ax es was calculated as the square root of the sum of squa of each axis. The acceleration at the threshold was calculated by scaling the acceleration measured at no attenuation by the attenuation at threshol d. For sinu signals, particle velocity is the particle acceleration divided by 2f, and particle displacement is particle velocity divided by 2 f. The sensitivity of the accelerometer was verified by comparing its output when di rectly vibrated with t he output of a lase vibrometer pointed at the accelerometer (Polytec, CLV 1000, Irvine, CA, USA). The laser vibrometer could not be used in t he manatee tank because it only measures motion in one direction along the laser beam As the research progressed, six underwater hydrophones (HTI-96MIN, Gulfport, MS, USA; s ensitivity -164 dBV/Pa; 2 Hz-37 kHz) arrayed on each face of a cube, 2 on each axial plane (20 cm apart), wer used to measure pressure gradients of the stimulus as well as monitor any no generated by the equipment. To calculate the pressure gr adient, dipole signals recorded simultaneously on all hydrophones. Pressure signals from each pair phones representing the th ree axes (X, Y, and Z), were subtracted and divided b the distance between them to calculate pre ssure gradient. The pressure gradient was divided by the water density to estimate the particle acceleration. For sinusoidal signals, the particle velocity, acceleration, and displacement were calculated using the same formulas as with the acceleromete r measurements. A ll measurements are presented as the magnitude of t he three directions calculated as the square root of th sum of each direction squared. 54

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To ensure that the test subjects w ere not cued during testing, a number of protocols and measurements were conducted. A 3-D accelerometer was routinely attached to the stationing apparatus to ensur e that there was no vi brational tra from the shaker during presentat ion trials. The research trainer responsible for verifying the position of the manatee and providing the primary reinforcement was blind to whether the ensuing trial was a stimulus-present or stimulus-absent trial. This trainer was also out of the manatees direct line of sight and remained motionless until the tri sequence was complete. Two underwater laser pointers (Lasermate SL6505M, Camino De Rosa, CA, were attached to the shaker apparatus by ball mounts and positioned to converg cm inline with the center of the stimulus generating sphere (Figure 3-3). The laser locations were monitored via a subm ersible video camera (HelmetCamera, Fredricksburg, VA, USA) and re corded using a portable DVR unit (DTY Industrial, Guangdong, China). Experiment II Restriction Tests To determine if the vibrissae contributed to detection of the hy drodynamic stimuli, trials were conducted using the same pr ocedure as in Experim ent I with the only difference being the presence of a neoprene wrap. The manat ees were trained to a 2 mm neoprene wrap with a 15.24 cm x 15.24 cm square opening allowing for a small numbers of post-facial vibrissae to be exposed to the stimuli (Figure 3-4, 3-5). The threshold testing was conducted at four locati ons of the post-facial body: right-side front right-side mid, right-side rear, an nsfer al USA) e at 20 V5, wear d left-side front. 55

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Results Experiment I Tacto gram Results for the behavioral post-facial ta ctogram highlight the sensitivity and frequency dependence of the detection of hydrody namic stimuli (Table 3-1). The data were combined, allowing for a comparat ive presentation betw een the sensitivity thresholds of the facial, pos t-facial, and restricted post-faci al experiments. The best sensitivity for each frequency was present ed and the false alarm rate for each frequency was averaged for the 4 locations. T he data for the 4 locations (Tables 3-1, 32, 3-3 played r At 150 Hz Buffett detected particle displace ment near 1 nm using ely correlated with frequency, with an incre stimuli in com parison to the non-wrap condition (Table 3-6). Threshold values we re calculated in terms of displacement, velocity, and acceleration as it is unknown which parameter the manatees detect with 3-4) demonstrate the similar sensitivity of the ma natee across the body for the detection of the hydrodynamic stimuli. Thre shold values were calculated in terms of displacement, velocity, and acceleration as it is unknown wh ich parameter the manatees detect with their vibrissae (Figure 36, 3-7, 3-8). Both subjects dis thresholds below 1 micron of particle disp lacement for frequencies above 10 Hz, simila to the facial vibrissae. post-facial vibrissae. Sensitivity wa s positiv ase in sensitivity obser ved at higher frequencies. Both manatees demonstrated similar thresholds, suggesting that t he combined tactogram may be a reasonable representation of the abilities of manatees generally. Overall, the post-facial vibrissae appear to be slightly less sensitive than the facial vibrissae. Experiment II Restriction Tests Data from the restriction trials demonstr ated that the thresholds increased overall as fewer vibrissae (10 20) were exposed to the 56

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their vibrissae (Figure 3-9, 3-10, 3-11). Both subjects displayed thresholds be low 1 nt for frequenc ies above 35 Hz, elevated compared to the unres ve am g d to n facial t ues (2007a), there is a represent ative population of recep he micron of particle displaceme tricted post-facial vibrissae. The th resholds at all frequencies were well abo those compared to previous experiments on the manatees vibrissae sensitivity. Both manatees demonstrated similar thresholds, suggesting that the combined tactogr may be a reasonable representati on of the abilities of manat ees generally. The limitin of the exposure of the postfacial vibrissae to the hydrodynamic stimuli appeare have a significant effect in elevating the sensitivity. Discussion The sensitivity threshold of the post-facial vibrissae, although slightly elevated, demonstrates a remarkable similarity to t he data from the facial vibrissae. Whe compared, the thresholds of the vibrissae demonstrate an increase progressing from the facial region to the post-facial region; however the levels are reasonably comparable (Figure 3-12, 3-13). As the number of post-facial vibriss ae exposed to the stimuli is reduced, the thresholds increased, in a manner similar to results observed for the vibrissae. The BLHs may be intermediate FS Cs, comprised of anatomical features tha suggest a role in both active and passive tact ile sensitivity. T he greater density of vibrissae on the facial region may account for the increased sensitivity demonstrated there (Reep et al., 1998; 2002). As shown by Sarko and colleag tor types associated with each follicle cla ssification. As the modality shifts from a predominance of active touch (facial vibris sae) to passive detection (post-facial vibrissae), there appears to be a transition of receptors and associated axons with t BLHs possessing an intermediate population and number, being involved in both 57

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detection scenarios. The presence of a highly developed somatosensory system is apparent in the neural architecture. T here is prominent representation of somatosensation in the brainstem and thalam us that appears to r epresent the fluke, flipper, tactile hairs of the post-facial body, pe rioral face, and the oral disk, from which a thalamic map (regionalized repres entations of specific areas of the body associated w specific loc ations in the brain) of the ani mal was derived (Sarko et al., 2007a). The presumptive somatosensory cortex is more ex tensive than the auditory or visual cort and represents ~25% of the total cortical area. Cort ith ex, ical representations of the postfacial hairs are hypothesized to be rep he small Rindenkerne in area CL2 partic of the it y slightly higher at 100 Hz. The blind cavefish might provi roduce resented by t ularly. Rindenkerne are neuronal aggregations found in layer 6 in five cortical areas may be similar to the somatosensory barre ls of other taxa. A large amount brainstem, thalamus, and cortex appears devoted to processing somatosensory information (Reep et al., 1989, Marshall et al ., 1995, Reep et al., 2002, Sarko et al., 2007b). Behavioral studies with several species of fish have demonstrated comparable results to manatee thresholds. Oscars ( Astronotus ocellatus ), goldfish ( Carassius auratus ), and toadfish ( Opsanus tau ) displayed particle displacement detection thresholds near or less than 1 nm (RMS) (Fay and Olsho, 1979; Fay, 1984; Fay et al., 1994) with the manatees sensitiv de a more direct applicable comparison as it utilizes self-produced hydrodynamic stimuli to detect objects as they near or pass them (Cam penhausen et al., 1981; Weissert and Campenhausen, 1981; Hassan, 1989). Objects in aquatic media p a boundary layer and the generate turbulence when introduced in flow fields, and 58

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manatees may be able to detect these perturbations an d utilize them as orientation and/or navigational cues. The ability of the manatee to detect hydrodynamic stimuli below a micron and down to a nanometer highlights the likelihood t hat manatees utilize their tactile sense to navigate through the often turbid waters w here they are found. The vibrissae of manatees are anatomically specialized and behaviorally utilized to detect hydrodyna stimuli, supporting and strengt hening the hypothesis that the vibrissae act as a sensory array analogous to the latera l line system of the fish. mic 59

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Table 3-1. Post-facial threshold values fo r each tested frequency for Buffett and Hug h. Buffett Frequency Displacement (Hz) Veloc ity Acceleration False Alarm ( m) (mm/s) (mm/s2) Rate 5 2.1131 0.0664 2.0856 0.12 10 1.5236 0.0957 6.0148 0.14 100 0.0149 0.0094 5.8779 0.14 50 0.0013 0.0012 1.1728 0.15 15 0.3679 0.0347 3.2676 0.11 25 0.1064 0.0167 2.6256 0.14 75 0.0066 0.0031 1.4765 0.17 125 0.0031 0.0024 1.9021 0.14 1 Table 3-1. Continued. Hugh Frequency (Hz) Displacement ( m) Velocity (mm/s) Acceleration (mm/s2) False Alarm Rate 5 5.0110 0.1574 4.9457 0.14 10 2.5578 0.1607 10.0977 0.11 15 0.4372 0.0412 3.8835 0.03 25 0.7112 0.1117 17.5479 0.08 75 0.0315 0.0148 6.9859 0.08 100 0.0105 0.0066 4.1613 0.06 125 0.0073 0.0057 4.5105 0.08 150 0.0062 0.0059 5.5491 0.15 60

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Table 3-2. Post-facial threshold values for the right-side front location for each tested frequency for Buffett and Hugh. Buffett Frequency z) acement ( (m tion (2Alarm R (H Displ m) Velocity m/s) Accelera mm/s ) False ate 5 2.5065 0.0787 2.4738 0.06 10 1.5236 0.0957 6.0148 0.14 25 0.1064 0.0167 2.6256 0.08 75 0.0066 0.0031 1.4765 0.06 100 0.0156 0.0098 6.1543 0.21 125 0.0031 0.0024 1.9021 0.13 Table 3-2. Contin ugh ued. H Frequency isplac (Hz) 5 D ement eloc ity cceleration 2alse Alarm 27 ( m) 5.21 V (mm/s) 0.1638 A (mm/s ) 5.1447 F Rate 0.06 10 10.5178 2.6642 0.1674 0.09 25 0.7112 0.1117 17.5479 0.04 75 0.0315 0.0148 6.9859 0.07 100 0.0107 0.0067 4.2107 0.10 125 0.0079 0.0062 4.8613 0.08 150 0.0066 0.0062 5.8347 0.20 61

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Table 3-3. Post-facial threshold values fo r the right-side mid location for each tested frequency for Buffett and Hugh. Buffett Frequency (Hz) Displacement ( m) Velocity (mm/s) Acceleration (mm/s2) False Alarm Rate 5 2.1131 0.0664 2.0856 0.12 10 1.6300 0.1024 6.4348 0.16 15 0.3679 0.0347 3.2676 0.11 25 0.1165 0.0347 2.8756 0.25 75 0.0067 0.0032 1.4868 0.20 Table 3-3. Continued. Hugh Frequency isplacement eloc ity cceleration alse Alarm 10 (Hz) 5 D ( m) 5.01 V (mm/s) 0.1574 A (mm/s2) 4.9457 F Rate 0.18 10 10.0977 2.5578 0.1607 0.15 15 0.4372 0.0412 3.8835 0.03 25 0.8020 0.1260 19.7884 0.16 75 0.0316 0.0149 7.0110 0.13 62

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Table 3-4. Post-facial threshold values fo r the right-side rear location for each tested frequency for Buffett and Hugh. Buffett Frequency (Hz) Displacement ( m) Velocity (mm/s) Acceleration (mm/s2) False Alarm Rate 75 0.0067 0.0032 1.4977 0.18 100 0.0149 0.0094 5.8779 0.21 125 0.0032 0.0025 1.9877 0.25 150 0.0013 0.0012 1.1728 0.10 Table 3-4. Continued. Hugh Frequency isplacement eloc ity cceleration alse Alarm 28 (Hz) 75 D ( m) 0.03 V (mm/s) 0.0155 A (mm/s2) 7.2858 F Rate 0.00 100 0.0105 0.0066 4.1613 0.03 125 0.0073 0.0057 4.5105 0.03 150 0.0062 0.0059 5.5491 0.12 63

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Table 3-5. Post-facial threshold values for the left-side front location for each tested frequency for Buffett and Hugh. Buffett Frequency (Hz) Displacement ( m) Velocity (mm/s) Acceleration (mm/s2) False Alarm Rate 5 2.4960 0.0784 2.4635 0.17 10 1.6546 0.1040 6.5322 0.12 25 0.1238 0.0194 3.0540 0.09 75 0.0069 0.0032 1.5247 0.24 100 0.0154 0.0097 6.0730 0.00 5 0.0027 2.1446 0.04 125 0.003 150 0.0015 0.0014 1.3455 0.20 Table 3-5. Cont. h inued Hug Frequency Displacement ( V (m Acceleration ( False Alarm R (Hz) m) elocity m/s) mm/s2) ate 5 5.2127 0.1638 5.1447 0.18 10 2.6642 0.1674 10.5178 0.08 5 0.8020 0.1260 19.7884 0.13 5 0.0325 0.0153 7.2115 0.10 25 0.0077 0.0060 4.7364 0.13 50 0.0075 0.0071 6.6648 0.13 2 7 1 1 64

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Table 3-6. Post-facial threshold with neoprene wrap values for each tested frequency for Buffett and Hugh. Buffett Frequency (Hz) Displacement ( m) Velocity (mm/s) Acceleration (mm/s2) False Alarm Rate 10 1.5992 0.1005 6.3133 0.05 15 2.2044 0.2078 19.5809 0.0119 6.3467 0.14 0.0251 0.0158 9.9151 0.21 05 0.11 25 1.9482 0.3060 48.0692 0.24 35 1.4298 0.3144 69.1460 0.10 50 0.3477 0.1092 34.3164 0.12 55 0.3419 0.1182 40.8341 0.08 75 0.0716 0.0337 15.8965 0.06 85 0.0223 100 125 150 0.0286 0.02 0.0225 0.0193 17.6564 18.1725 0.07 0.13 Tab Hugh le 3-6. Conti nued. Frequency (Hz) Displacement ( Veloc (mm/s) Acceleration (mm/s False Alarm Rate 0.1333 m) ity 2) 10 2.1218 8.3767 0.21 15 2.2044 0.2078 19.5809 0.4679 102.8992 0.08 0.1563 49.0996 0.10 5 0.7121 0.2461 85.0429 0.00 0.0440 20.7255 0.08 0.0268 21.0121 0.07 50 0.0393 0.0371 34.9449 0.20 0.04 35 2.1277 50 0.4975 5 75 0.0933 125 0.0341 1 65

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Figure 3-1. Mantee at stati on, prepared for a test trial on the post-facial vibrissae. cat e padd o (Photos a Lo courtesy of author) ion of shaker and respons le to the right f the manatee 66

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Figure 3-2. Diagram showing the 4 locations tested during the vibr otactile tactogram. 67

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Figure 3-3. Shaker set-up with waterproof housing, attached lasers (gold), and camera (top of aluminum mount). (Photos courtesy of author) 68

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69 Figure 3-4. Manatee undergoing training to h abituate to wearing the neoprene wrap. (Photos courtesy of author)

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70 Figure 3-5. Manatee at station, prepared for a test trial on t he post-facial vibrissae while wearing the neoprene wrap. Location of shaker and response paddle to the right of the manatee. No te the square opening in the neoprene wrap. (Photos courtesy of author)

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0.001 0.01 0.1 1 10 1101001000Frequency (Hz)Displacement ( m) Buffett Hugh Figure 3-6. Threshold values for displaceme post-facial vibrissae for both te st subjects. Buffett (solid diamond, solid line) and Hugh (open circle, dashed line). Both the x-ax is and y-axis are represented with logarithmic scales. nt detection by 71

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0.001 0.01 0.1 1 1101001000Frequency (Hz)Velocity (mm/s) Buffett Hugh Figure 3-7. Threshold values for velocity detection by post-facial vibrissae for both test subj ects. Buffett (solid diamond, solid line) and Hugh (open circle, dashed line). Both the x-axis and y-axis are represented with logarithmic scales. 72

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73 1 10 100 1101001000Frequency (Hz)Acceleration (mm/s2) Buffett Hugh Figure 3-8. Threshold values for acceleration detection by pos t-facial vibrissae for both test subjects. Buffett (solid diamond, solid line) and Hugh (open circle, dashed line). Both the x-ax is and y-axis are represented with logarithmic scales.

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0.01 0.1 1 10 1101001000Frequency (Hz)Displacement ( m) Buffett Hugh ffett Figure 3-9. Threshold values for displacement detection by re stricted post-facial vibrissae fo r both test subjects. Bu (solid diamond, solid line) and Hugh (open circle, dashed line). Both the x-axis and y-axis are r epresented with logarithmic scales. 74

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0.01 0.1 1 1101001000Frequency (Hz)Velocity (mm/s) Buffett Hugh Figure 3-10. Threshold values for velocity detection by restri cted post-facial vibrissae for bot h test subjects. Buffett (sol id diamond, solid line) and Hugh (open circle, dashed line). Both the x-ax is and y-axis are represented with arithmic scales. log 75

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76 Figure 3-11. Threshold values for acceleration detection by rest ricted post-facial vibriss ae for both test subjects. Buffett (solid diamond, solid line) and Hugh (open circle, dashed line). Both the x-axis and y-axis are r epresented with logarithmic scales. 1 10 100 1000 1101001000Frequency (Hz)Acceleration (mm/s2) Buffett Hugh

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0.001 0.01 0.1 1 10 1101001000Frequency (Hz)Displacement ( m) Facial Post-facial Wrap Figure 3-12. Comparison of threshold va lues for displacement detection by Buffe tt. Both the x-axis and y-axis are represented with logarithmic scales. 77

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0.0001 0.001 0.01 0.1 1 10 1101001000Frequency (Hz)Displacement ( m) Facial Post-facial Wrap Figure 3-13. Comparison of threshold va lues for displacement detection by Hugh. Both the x-axis and y-axis are represented with logarithmic scales. 78

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CHA PTER 4 DETECTION OF DIRECTIONALITY OF HYDRODYNAMIC STIMULI BY THE POSTFACIAL VIBRISSAE OF THE FLORIDA MANATEE ( TRICHECHUS MANATUS LATIROSTRIS) Background Understanding the features of stimuli that manatees use to gain information about their environment would provide crucial insigh t into the ways in which the post-faci vibrissae are utilized as a sensory system. In a wild setting, there are likely to be multiple hydrodynamic cues generated at any given moment. Although it might be difficult to assess which cues are most important to manatees, we hypothesize tha post-facial hairs play a role in their ability to localize as well as detect hydrodynam stimuli based upon behavioral observations and the extensive neural investment manatees possess in the FSCs. Therefore, we construct ed a test of the ability of manatees to detect and localize hydrodynamic stimuli. Marine mammals have demonstrated the ab ility to detect hydrodynamic stim and track prey utilizing their vibrissae (Dehnhardt et al., 2001, Glaser et al., 2011). Tracking involves detection followed by the resolution of intensity differences and consequent adjustments in the direction of movement. Manatees may utilize simil cues to determine the presence of conspec ifics, obstacles, and currents in their environment. It is possible t hat different types of vegetat ions create distinctive flow patterns that manatees can discern. Materials and Methods Subjects The subjects were two male Florida manatees ( Trichechus manatus latirost housed at Mote Marine Laboratory & Aquarium in Sarasota, Florida, USA. Buffett and al t ic uli ar ris ) 79

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Hugh, 25 and 28 years of age, respec itiation of the study, had an exte t al., 2001; Bauer et al., 2003; Mann et al., 2005; Colbert et al., 2009; Bauer et al., 2012; Gaspard et al., 2012). The manatees were trained utilizing operant conditioning through positive reinforcement to signal the detection of hydrody namic stimuli directed at their post-facial vibrissae. A 2-choice (stimulus is pres ented on the left or right side) procedure with catch trials was used to determine stimul us detection. The testing procedure was modified from previous experiments to in clude a second shaker located on the opposite side of the subject and a side-specific response to the stimu li (Figure 4-1). The subject indicated the detection of the stimulus by withdrawing from the stationing apparatus and pressing a laterally positioned response paddle located on the same side as the shaker that generated the stimulus. Catch trials, defined operationally as remaining stationed for a ten second period after the initiation of a signal absent trial, were used on 25% of the total trials. Correct responses were followed by an auditory secondary reinforcer, a digitized whistle from an underwater speak er, followed by primary reinforcement, preferred food items of pieces of apples, carrots, beets, and monkey biscuits. Four warm-up trials (2 left side, 2 right side) were conducted prior to testing to assess the motivation and performance levels of the manatees with the stimulus at the same tested. A criterion of 75% correct on warm-up trials had to be met in order for testing to occur du tively, at the in nsive training history in the context of husbandry and sensory research (Colbert e Procedures frequency and level that was to be ring that particular session. If the subject failed to meet criterion on the first set of warm-up trials, a second warm-up set was 80

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conducted. Testing was not conducted if the subject failed to meet criterion on th second war m-up block. The subjects were trained to station by placing their postnasal crease on a horizontal PVC bar (2.5 e cm diameter) at a d epth of 0.75 m. T he manatees body was een the 2 shakers to ensure similar levels of t he stimulus(i) was received by ei r a n o the se, ere vibrational transfer between the shaker and t he manatee, the stationing apparatus and centered betw ther side. A tri-cluster LED signa led t he initiation of every trial, illuminating fo duration of 1 s, followed by a 0.5 s delay prior to both signal present and signal absent windows. The stimuli were generated by a 5.7 cm sinusoidally oscillating sphere drive by a computer-controlled calibrated vibratio n shaker. The sphere was connected t shaker via a rigid stainless steel rod. Ea ch shaker and attachment rod were oriented horizontally in the water column. The shak ers were housed in water-tight cylindrical housings with the rod passing through a sealed silicone barrier. The stimuli were 3 seconds in duration with cos2 rise-fall times of 300 ms and ranged from 25 125 Hz. Daily sessions (weekdays) were conducted with each session focused on a single frequency. An underwater speaker presented masking noise throughout the session to mask any auditory artifacts generated by the shaker. Equipment Two dipole vibration shak ers (Data Physics Signal Force, Model V4, San Jo CA, USA), each with a 5.7 cm diameter rubberized sphere connected via a rigid stainless steel extension rod, were posit ioned on either side of the manatee and w used to generate the stimuli fo r the directionality study. The dipole shaker generates a localized flow that decreases in amplitude as 1/distance3, as opposed to a monopole source that decreases in amplitude as 1/distance2 (Kalmijn, 1988). To eliminate any 81

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the shaker mount were separate pieces of equipment buffered with shock absorbing foam. The shakers were aligned t o direct the stimuli at the same location of the mana umn. ) 2 kHz bandwidth) constantly ession to mask any auditory artifacts from t he generation of the hydro r g. tee, though on differing sides which included the same depth in the water col Two identical hardware systems were designed, each serving a single vibration shaker. The stimuli were generated digitally by a Tucker-Davis Technologies (TDT) Enhanced Real-Time Processor (RP2.1, Alachua, FL, USA; sample rate 24.4 kHz), attenuated with a TDT Programmabl e Attenuator (PA5) to c ontrol level, and amplified with a Samson Power Amplifier (Servo 120a, Hauppauge, NY, USA). The signal generating equipment was controll ed by a program in MATLAB (MathWorks, Natick, MA, USA) in conjunction with a graphical us er interface (TDT Real-Time Processor Visual Design Studio) created specifically for this research. A digital output on an RP2.1 was used to control the LED that indicated the start of a trial. A separate D/A channel was used to generate the acoustic secondary reinforcer, which was presented through an underwater speaker (Clark Synthesis, Model AQ-39, Littleton, CO, USA when the manatee was correct on a trial. The speaker was located >1 m away from the subject and also presented noise (151 dB re 1 Pa; 12. through the s dynamic stimulus. These signals were amplified by a separate amplifier (American Audio, Model VLP 300, Los A ngeles, CA, USA) to avoid crosstalk. For stimuli analysis and calibration, a 3-dimensional accelerometer (Dimension Engineering, Model DE-ACCM3D Akron, OH, USA) was em bedded into the sphere to measure its movement. MATLAB was used to calculate, plot, and log the stimulus fo each trial. This accelerometer was used to monitor the shaker operat ion during testin 82

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To calculat e particle motion from the dipol e for threshold measur ements, six underwa hydrophones (HTI-96-MIN, Gulf port, MS, USA; sensitivity 164 dBV/Pa; 2 Hz-37 arrayed on each face of a cube, 2 on each axial plane (20 cm apart), were used to ter kHz) meas by the le hree ing to t trial. tionless until ure pressure gradients of the stimulus as well as monitor any noise generated by the equipment. To calculate the pressure gradient, dipole sign als were recorded simultaneously on all hydrophon es. Pressure signals from each pair of hydrophones representing the three axes (X, Y, and Z), were subtracted and divided by the distance between them to calculate pressure gradient. The pressure gradient was divided water density to estimate the particle accele ration. For sinusoidal signals, particle velocity is the particle acceleration divided by 2 f, and particle displacement is partic velocity divided by 2 f. All measurements are presented as the magnitude of the t directions calculated as the square root of the sum of each direction squared. To ensure that the test subjects were not cued during testing, a number of protocols and measurements were conducted. A 3-D accelerometer was routinely attached to the stationing apparatus to ensure that there was no vi brational transfer from the shaker during presentat ion trials. The research trainer responsible for verify the position of the manatee and providing the primary reinforcement was blind whether the ensuing trial was a stimulus-present (either side) or stimulus-absen This trainer was also out of the manatees direct line of sight and remained mo the trial sequence was complete. Two underwater laser pointers (Lasermate SL6505M, Camino De Rosa, CA, USA) were attached to a shaker apparatus by ball mounts. When the lase r points aligned, the subject was exactly 20 cm from the stimulus generating sphere and a trial was 83

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commenced. The laser locations were m onitored via a submersible video camera (HelmetCamera, Fredricksbur g, VA, USA) and recorded using a portable DVR unit (DTY Industrial, V5, Guangdong, China). Results Results for the behavioral directionality det ection of hydrodynamic stimuli by facial vibrissae demonstrate the ability of both manatees to determine the direction of the hydrodynamic stimuli at well above chance levels (Table 4-1). Both subjects correctly identified the directi on of the stimulus and responded to catch trials at 85% or above for all conditions. The false alarm per centages were very low, frequently zer highlighting the conservative strategy that both manatees appear to employ, consistent with previous studies. Both manatees demonstrated similar percentages, suggesting that these may be a reasonable representation of the abilities of manatees gen posto, erally. str or rooted in the benthic substrate. H owever, their Discussion The high percentage of correct responses for directionality detection by the po facial vibrissae clearly demonstrates t he manatees ability to perceive lateral hydrodynamic stimuli. Carnivorous aquatic mammals have demonstrated the ability to follow the hydrodynamic trails produced by pr ey species utilizing facial vibrissae, primarily mystacial (Dehnhardt et al., 2001, Glaser et al., 2011). As herbivores, manatees do not need the ability to track mobile prey items sinc e their forage is typically found floating at the surface of the wate ability to detect hydrodynamic stimuli may help them in locating forage areas and possibly even discriminating among different types of vegetation, such as algae versus seagrass. 84

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We hypothesize that hydrodynamic stimuli are most important to manatee migration and local orientati on. Manatees migrate biannually between warm water winter refugia and locations with abundant v egetation during the summer. Manatees spend a significant portion of their time iaters and it is not known what cues mana fresh water sources, typically up rivers to consume freshwater vegetation and d ironmental llow ebb y fish, manatees may utilize thei r passive sense of touch to determine the n turbid w tees use during migrati on or for orientation duri ng shorter-range transits. Manatees possess poor visual acuity (Mass et al., 1997; 2012; Bauer et al., 2003) and do not echolocate. As demonstrated by pr evious research, manatee vibrissae are highly sensitive. With the presence of ~5,300 vibrissae, there is a significant central nervous system representation and dedication to the tactile modality. The manatees ability to determine the direction of hydr odynamic stimuli begins to demonstrate a mechanism for receiving cues that would a llow them to deftly swim through complex environments. Manatees travel repeatedly between coastal, high-salinity locations to and creeks, rink fresh water (Stith et al., 2006). As manatees swim against a current, the directional flow provides an abundance of hydr odynamic cues, as well as env cues such as differences in temperature or salinity. For example, turbulence created by in-water objects may cue manatees to avoid them. Manatees can be found in sha areas vulnerable to extremely low tides, such as coastal creeks in Georgia. The movement of the out-going ti de might serve as an indicator for the manatee to move back downstream before becoming stranded (Zoodsma, 1991). During winter months manat ees congregate at warm water refugia coming into contact with a greater density of conspecifics than at othe r times of the year. As displayed b 85

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move erved e ch other e of ment of conspecifics and maneuver a ccordingly. Manatees have been obs to breathe synchronously, especially during periods of rest, without any visual or auditory communication. Although it is not known what state of sleep manatees may b in during this process, t he only prompt available would appear to be tactile via hydrodynamic stimuli. Manatees have been observed to initiate contact with ea by actively using their facial vibris sae on the lower dorsal region of a conspecific. Further research is necessary to test t hese hypotheses regarding the manatees us the vibrissal array. 86

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Table 4-1. Percentage correct on directiona lit y test trials based on the presentation of The average displacement values of the stimulus from both shakers for Buffett the stimuli directed at the s ubjects left or right side tr ials and false alarm rate. each frequency is also presented. Frequency (Hz) Left Right False AlarmDisplacement ( m) 25 100 100 0 2.4365 50 100 85 15 0.4091 75 100 95 0 0.1498 125 100 100 0 0.0431 Table 4-1. Continued. Hugh Frequency (Hz) Left Right False AlarmDisplacement ( m) 25 100 100 0 2.4365 50 94 88 0 0.4091 75 86 100 14 0.1498 125 100 100 0 0.0431 87

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Figure 4-1. Manatee at station, prepared for a test trial of di rectionality detection by the post-facial vibrissae. Location of shakers and response paddles are equidistant to either side of the manatee. 88

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CHA PTER 5 CONCLUSION Florida manatees primarily i nhabit the coastal and inland wa ters of the peninsular state. Manatees seem to have little difficu lty navigating these turbid waterways which often contain obstacles that they must maneuver around. Manatees likely use their tactile sense to detect currents, tidal movem ents, in-water obstacles, and to facilitate interaction with conspecifics. The hydrody namic stimuli detection thresholds of the facial and post-facial vibrissae demonstrat ed similar loss of sensitivity when their exposure was restricted by the mesh netting (facial vibrissae) or limited by the neoprene wrap (post-facial vibrissae). We c onclude, based on anatomical and behavioral psychophysical testing, manatees are somatos ensory specialists. Furthermore, due to the similar nature of the stim ulus and frequencies, it seems plausible to suggest that the manatees vibrissae serve as a mammalian lateral line. Significance Manatees are an endangered species, so it is important to consider the conservation implications of this research. As tactile specialists, manatees may have a limited ability to adapt to human-caused cha nges to their environment. Hydrodynamic situations. The close proxim moving boats, closing flood and lock gates, and monofilament fishing gear limits the potential response time to avoid such hazards. It is possible that manatees can detect the bow waves of slow moving boats and avoid a collisi on, highlighting the utility of slow speed zones (Calleson and Frohlich, 2007), especially where hydrodynamic stimuli may be distorted, such as in shallow seagrass beds. The vibrissae of manatees are anatomically specialized and stimuli attenuate rapidly, pr oviding little reaction time in the event of dangerous ity required for the tactile detection of 89

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behaviorally utilized to detect hydrody supporting and strengthening the hypothesis that the vibrissae act as a sens ory array analogous to the lateral line system of the fish. namic stimuli, 90

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Dehnhardt, G., Mauck, B., Hyvri nen, H. 1998. Ambient te mperature does not affect th tactile sensitivity of mystacial vibrissae in harbour seals. J Exp Biol 201: 3023 3029. e Ebara, S., Kumamoto, K ., Matsuura, T., Ma zurkiewicz, J.E., Rice, F.L. 2002. Similarities d cat: a Fay, F.H. 1982. Ecology and bi ology of the Pacific walrus, Odobenus rosmarus Fay, R.R. 1984. The goldfish ear codes t he axis of acoustic particle motion in three Fay, cular afferents of the toadfish to linear acceleration at audio frequencies. Biol Bull 187: Fay, R.R., Olsho, L.W. 1979. Discharge patterns of lagenar and saccular neurons of J Exp Biol, 215: 1442-1447. 5Glaser, N., Wieskotten, S., Otter, C., Dehnhard t, G., Hanke, W. 2011. Hydrodynamic Hanke, W., Witte, M., Miersch, L., Brede, M., Oeffner J., Michael, M., Hanke, F., Leder, A., Dehnhardt, G. 2010. Harbor seal vi brissa morphology suppresses vortexHartman, D.S. 1979. Ecology and behavior of the manatee ( Trichechus manatus ) in Dykes, R.W. 1975. Afferent fibers from mystacial vibrissae of cats and seals. J Neurophysiol. 38: 650-662. and differences of mystacial vibrissal follicle-sinus complexes in the rat an confocal microscopic study. J Comp Neurol 449:103-119. divergens USFWS, North Americ an Fauna, 74. Wash. DC. dimensions. Science, 225: 951-954. R.R., Edds-Walton, P.L. Highstein, S.M. 1994. Directional sensitivity of sac 258-259. the goldfish eighth nerve: Displacement sensitivity and directional characteristics. Comp Biochem Physiol, 62: 377-386. Gaspard, J.C. III, Bauer, G.B. Reep, R.L., Dziuk, K., Cardwell, A., Read, L., Mann, D.A. 2012. Audiogram and auditory critical ratios of two Florida manatees ( Trichechus manatus latirostris ). Gerstein, E.R., Gerstein, L., Forsythe, S., Blue, J. 1999. The underwater audiogram of the West Indian manatee (Trichechus manatus). J Acoust Soc Am 105: 357 3583. Gescheider, G.A. 1997. Psychophysics: the fundamentals. Lawrence Ehrlbaum Associates, Mahwah, NJ. trail following in a California sea lion ( Zalophus californianus ). J Comp Physiol A 197: 141-151. induced vibrations. J Exp Biol 213: 2665-2672. Florida. Am Soc Mamm Special Pub, 5: 1-153. 93

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Hassan, E.S. 1989. Hydrodynam ic imaging of the surroundings by the lateral line of the blind cave fish Anoptic hthys jordani In Coombs, S., Grner, P., Mnz, H. ed. T Mecha he nosensory Lateral Line, Neurobiology and Evolution, Springer-Verlag, New York, 217. Hyvrinen, H. 1995. Structure and function of the vibrissae of the ringed seal ( Phoca hispida L.) In Kastelein, R.A., Thomas, J.A ., Nachtigall, P.E. ed. Sensory systems Kalm In Atema, J., Fay, R.R., Popper, A.N., Tavolga, W.N., eds. Sensory Biology of Aquatic Animals, Kastelein, R.A., Van Gaalen, M.A. 1988. The tactile se nsitivity of the mystacial r, ysiol 191: 903-908. Rec 288A: 13-25. e ( Trichechus manatus latirostris ). Mar Mamm Sci 14: 274-289. s manatus latirostris Brain Behav Evol, 45: 1-18. okl of aquatic mammals, De Spil Pub lishers, The Netherlands, 429-445. ijn, A. J. 1988. Hydrody namics and acoustic field detec tion. Springer-Verlag, New York, 83. vibrissae of a Pacific walrus (Odobenus rosmarus divergens ). Part 1. Aquatic Mamm, 14: 123-133. Lu, Z., Popper, A.N., Fay, R.R. 1996. Behavioral detection of acoustic particle motion by a teleost fish ( Astronotus ocellatus): sensitivity and directionality. J Comp Physiol A 179: 227-233. Mann, D.A., Colbert, D.E., Gas pard, J.C., Casper, B.M., C ook, M.L., Reep, R.L., Baue G.B. 2005. Temporal resoluti on of the Florida manatee ( Trichechus manatus latirostris ) auditory system. J Comp Ph Marshall, C.D., Amin, H., Kovacs, K.M., Ly dersen, C. 2006. Microstructure and innervation of the mystacia l vibrissal follicle-sinus complex in bearded seals, Erignathus barbatus (Pinnipedia: Phocidae). Anat Marshall, C.D., Huth, G.D., Edmonds, V.M., Halin, D.L., Reep, R.L. 1998. Prehensile use of perioral bristles during feeding and associated behaviors of the Florida manate Marshall, C.D., Reep, R.L. 1995. Manatee cerebral cortex : cytoarchitecture of the caudal region in Trichechu Mass, A.M., Ketten, D.R., Ode ll, D.K., Supin, A.Y. 2012. Ganglion cell distribution and retinal resolution in the Florida manatee, Trichechus manatus latirostris Anat Rec 295: 177-186. Mass, A.M., Odell, D.K., Ketten, D.R., S upin, A.Y. 1997. Gang lion layer topography and retinal resolution of the Caribbean manatee ( Trichechus manatus latirostris). D Biol Sci 355: 392-394. Morley, J.W., Goodwin, A.W., Darian-Smith, I. 1983. Tactile discrimination of gratings. Exp Brain Res 49: 291. 94

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Reep, R.L., Johnson, J.I., Swit zer, R.C., Welker, W.I. 1989 Manatee cerebral cortex: cytoarchitecture of t he frontal regi on in Trichechus manatus latirostris Brain Behav Evol 34: 365-386. Reep, R.L., Marshall, C.D., Stoll, M.L., Whit aker, D.M. 1998. Distribution and s Reep, R.L., Marshall, C.D., Stoll, M.L. 2002. Tactile hairs on the postcranial body in Reep er, B.L. Samuelson, D.A. 2001. Microanatomy of facial vibrissae in the Fl orida manatee: the basis for specialized Rice, iddson, J. Aldskogius, H., Johansson, O. 1997. Comprehensive immunoflorescence and lectin binding analysis of vibrissal follicle Rice, F.L., Mance, A., Munger, B.L. 1986. A comparative li ght microscopic analysis of Sarko, D.K., Johnson, J.I., Switzer, R. C., III, Welker, W.I., Reep, R.L. 2007b. 90: Sarko, D.K., Reep, R.L., Mazurkiewicz, J.E ., Rice, F.L. 2007a. Adaptations in the l, xp Biol, 210: 781-787. Soko s. Mammalia 51: 125-138. Zelena, J. 1994. Nerves and Mechanorec eptors. Chapman and Hall, London. innervation of facial bristles and hairs in the Florida manatee ( Trichechus m anatu latirostris ). Mar Mamm Sci 14: 257-273. Florida manatees: a mammalian lateral line? Brain Behav Evol 59: 141-154. R.L., Stoll, M.L., Ma rshall, C.D., Hom sensory function and oripulation. Brain Behav Evol 58: 1-14. F.L., Fundin, B.T., Arv sinus complex innervation in the mystacial pad of the rat. J Comp Neurol, 385: 149-184. the sensory innervation of t he mystacial pad. I. Innervation of the vibrissal folliclesinus complexes. J Comp Neurol 252: 154-174. Somatosensory nuclei of the manatee brainstem and thalamus. Anat Rec 2 1138-1165. structure and innervation of follicle-sinus complexes to an aquatic environment as seen in the Florida manatee ( Trichechus manatus latirostris ). J of Comp Neuro 504: 217-237. Schulte-Pelkum, N., Wieskotten, S., Hanke, W., Dehnhardt, G., Mauck, B. 2007. Tracking of biogenic hydrodynamic trails in harbour seals ( Phoca vitulina ). J E lov, V.E., Kulikov, V.F. 1987. The structure and function of th e vibrissal apparatus in some rodent Stith, B.M., Slone, D.H., Re id, J.P. 2006. Review and synthesis of manatee data in Everglades National Park. USGS Administ rative Report. USGS Florida Integrated Science Center, Gaine sville, FL. 126 pp. Urick, R.J. 1983. Principl es of underwater sound, 3rd ed. McGraw-Hill Book Co., NY. 95

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96 Zoodsma, B.J. 1991. Distribution and behaviora l ecology of manatees in southeastern Georgia. M.S. Thesis, University of Florida, Gainesville, FL.

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BIOGRAPHICAL SKETCH Joe Gaspard was born in Newburgh, New York and raised in the seasonal Hudson Valley. Yearly vacations to the beach seeded his desir e to follow a path to become a marine biologist. After graduating fr om Newburgh Free Academy, he attended Southampton College Long Island University. During his time there, his desire to learn everything he could about anything aquatic was ignited. Ironically, one species that he did not know anything about was t he Florida manatee and Joe accepted an internship at a recently expanded Mote Marine Laboratory & Aquarium in Sarasota, Florida to work with them. He joi ned the fledgling Manatee Care and Research Department and was taught a strong and unique background of research training with a novel species. Working through the manat ees poor to extremel y sensitive sensory processes, new knowledge about this endangered species was gained. Joe was very fortunate to be able to work with a wide range of aquatic, terrestrial, and aquatic species after meeting amazing indivi duals not unlike himself at conferences and meetings focused on training and research. Not only di d he grow professionally through working with manatees but also personally. His fu ture wife was just as passionate about working with marine life that she was duped by Joe into cleaning the manatee tank. A number of years and two beautiful amazing children later, academically culminating in a Ph.D. from the University of Florida in the spring of 2013, the next chapter is just beginning. 97