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The functions of sound production in the lined seahorse, Hippocampus erectus, and effects of loud ambient noise on its b...

Permanent Link: http://ufdc.ufl.edu/UFE0024776/00001

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Title: The functions of sound production in the lined seahorse, Hippocampus erectus, and effects of loud ambient noise on its behavior and physiology in captive environments
Physical Description: 1 online resource (190 p.)
Language: english
Creator: Anderson, Paul
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acoustic, aquaculture, aquarium, courtship, feeding, foraging, hearing, hippocampus, masking, noise, prey, seahorse, sound, stress
Forest Resources and Conservation -- Dissertations, Academic -- UF
Genre: Fisheries and Aquatic Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: THE FUNCTIONS OF SOUND PRODUCTION IN THE LINED SEAHORSE, HIPPOCAMPUS ERECTUS, AND EFFECTS OF LOUD AMBIENT NOISE ON ITS BEHAVIOR AND PHYSIOLOGY IN CAPTIVE ENVIRONMENTS By Paul August Anderson August 2009 Chair: William J. Lindberg Major: Fisheries and Aquatic Sciences Loud noise in aquaria represents a cacophonous environment for captive fishes. I tested the effects of loud noise on acoustic communication, feeding behavior, courtship behavior, and the stress response of the lined seahorse, Hippocampus erectus. Total Root Mean Square (RMS) power of ambient noise to which seahorses are exposed in captivity varies widely but averages 126.1 +/- 0.8 deciBels with reference to one micropascal (dB re: 1 microPa) at the middle of the water column and 133.7 +/- 1.1 dB at the tank bottom, whereas ambient noise in the wild averages 119.6 +/- 3.5 dB. Hearing sensitivity of H. erectus, measured from auditory evoked potentials, demonstrated maximum spectrum-level sensitivities of 105.0 +/- 1.5 dB and 3.5 X 10-3 +/- 7.6 X 10-4 m/s2 at 200 Hz; which is characteristic of hearing generalists. H. erectus produces acoustic clicks with mean peak spectrum-level amplitudes of 94.3 +/- 0.9 dB at 232 +/- 16 Hz and 1.5 X 10-3 +/- 1.9 X 10-4 m/s2 at 265 +/- 22 Hz. Frequency matching of clicks to best hearing sensitivity, and estimates of audition of broadband signals suggest that seahorses may hear conspecific clicks, especially in terms of particle motion. Behavioral investigations revealed that clicking did not improve prey capture proficiency. However, animals clicked more often as time progressed in a courtship sequence, and mates performed more courtship behaviors with control animals than with muted animals, lending additional evidence to the role of clicking as an acoustic signal during courtship. Despite loud noise and the role of clicking in communication, masking of the acoustic signal was not demonstrated. Seahorses exposed to loud noise in aquaria for one month demonstrated physiological, chronic stress responses: reduced weight and body condition, and increased heterophil to lymphocyte ratio. Behavioral alterations were characterized by greater mean and variance of activity among animals housed in loud tanks in the first week, followed by habituation. By week four, animals in loud tanks demonstrated variable performance of clicking and piping, putative distress behaviors. Despite the physiological stress response, animals in loud tanks did not reduce feeding response or courtship behavior, suggesting allostasis.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Paul Anderson.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Lindberg, William J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024776:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024776/00001

Material Information

Title: The functions of sound production in the lined seahorse, Hippocampus erectus, and effects of loud ambient noise on its behavior and physiology in captive environments
Physical Description: 1 online resource (190 p.)
Language: english
Creator: Anderson, Paul
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acoustic, aquaculture, aquarium, courtship, feeding, foraging, hearing, hippocampus, masking, noise, prey, seahorse, sound, stress
Forest Resources and Conservation -- Dissertations, Academic -- UF
Genre: Fisheries and Aquatic Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: THE FUNCTIONS OF SOUND PRODUCTION IN THE LINED SEAHORSE, HIPPOCAMPUS ERECTUS, AND EFFECTS OF LOUD AMBIENT NOISE ON ITS BEHAVIOR AND PHYSIOLOGY IN CAPTIVE ENVIRONMENTS By Paul August Anderson August 2009 Chair: William J. Lindberg Major: Fisheries and Aquatic Sciences Loud noise in aquaria represents a cacophonous environment for captive fishes. I tested the effects of loud noise on acoustic communication, feeding behavior, courtship behavior, and the stress response of the lined seahorse, Hippocampus erectus. Total Root Mean Square (RMS) power of ambient noise to which seahorses are exposed in captivity varies widely but averages 126.1 +/- 0.8 deciBels with reference to one micropascal (dB re: 1 microPa) at the middle of the water column and 133.7 +/- 1.1 dB at the tank bottom, whereas ambient noise in the wild averages 119.6 +/- 3.5 dB. Hearing sensitivity of H. erectus, measured from auditory evoked potentials, demonstrated maximum spectrum-level sensitivities of 105.0 +/- 1.5 dB and 3.5 X 10-3 +/- 7.6 X 10-4 m/s2 at 200 Hz; which is characteristic of hearing generalists. H. erectus produces acoustic clicks with mean peak spectrum-level amplitudes of 94.3 +/- 0.9 dB at 232 +/- 16 Hz and 1.5 X 10-3 +/- 1.9 X 10-4 m/s2 at 265 +/- 22 Hz. Frequency matching of clicks to best hearing sensitivity, and estimates of audition of broadband signals suggest that seahorses may hear conspecific clicks, especially in terms of particle motion. Behavioral investigations revealed that clicking did not improve prey capture proficiency. However, animals clicked more often as time progressed in a courtship sequence, and mates performed more courtship behaviors with control animals than with muted animals, lending additional evidence to the role of clicking as an acoustic signal during courtship. Despite loud noise and the role of clicking in communication, masking of the acoustic signal was not demonstrated. Seahorses exposed to loud noise in aquaria for one month demonstrated physiological, chronic stress responses: reduced weight and body condition, and increased heterophil to lymphocyte ratio. Behavioral alterations were characterized by greater mean and variance of activity among animals housed in loud tanks in the first week, followed by habituation. By week four, animals in loud tanks demonstrated variable performance of clicking and piping, putative distress behaviors. Despite the physiological stress response, animals in loud tanks did not reduce feeding response or courtship behavior, suggesting allostasis.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Paul Anderson.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Lindberg, William J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024776:00001


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1 THE FUNCTIONS OF SOUND PRODUCT ION IN THE LINED SEAHORSE, HIPPOCAMPUS ERECTUS, AND EFFECTS OF LOUD AM BIENT NOISE ON ITS BEHAVIOR AND PHYSIOLOGY IN CAPTIVE ENVIRONMENTS By PAUL AUGUST ANDERSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Paul August Anderson

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3 To my parents, Josephine J. McLaughlin and Paul F. Anderson, whose unconditional love and support have paved the way for me to be everything that I want to be

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4 ACKNOWLEDGMENTS First and forem ost, I owe a debt of gratit ude to my Ph.D. advisor, W.J. Lindberg (University of Florida/UF), w ho accepted the daunting role of advising a student with complex, multidisciplinary research interests. Among th e many contributions made to the project, Dr. Lindberg provided wisdom and teachings in the scientific process, helped guide complex negotiations among many stakeholders involved, o ffered funding via the D.M. Smith Fellowship, and assisted in the acquisition of additiona l funds along the way. Dr. Lindberg provided enlightened guidance all along the route of this circuitous adve nture to its destination! Similarly, I offer many thanks to each of my Ph.D. committee members, I. Berzins (The Florida Aquarium) H. Masonjone s (University of Tampa), D. Mu rie (UF), D. Parkyn (UF), C. St. Mary (UF), each of whom played important roles in shaping the project into its final form. In particular, I thank I. Berzins for offering a fruitful and collaborativ e relationship with The Florida Aquarium Center for Conservation, and for generating ideas about the effects of noise on stress in fishes (that led to co-authorship in Chapter 4). I. Berzins also provided funding to conduct The Seahorse Sound Survey (Chapter 2) a nd has created a position for me at The Center for Conservation that has to date enabled me to fulfill mutual career goals; it is an opportunity that I truly cherish and enjoy. The concept of th e Dissertation was developed in consult with H. Masonjones, who first suggested effects of noise on seahorse health and reproduction, having previously detected effects in her study system (H. zosterae) and suggesting further exploration of the phenomenon H. Masonjones also captained the vessel (and furnished a crew of competent shipmates, S., G., and K. Masonjones) upon which ambient noise measurements were taken from the field. D. Murie provided thorough critique of study design and also suggested the study of acoustic roles in prey cap ture behavior, that led to Chap ter 5 of this Dissertation. D. Parkyn offered wisdom and teachings in the co mplex world of acoustic neuroethology, and was

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5 instrumental in securing stipend funding along the way. Finally, C. St. Mary also offered valuable critique of study design, assisted with fund acquisition, and advi sed statistical methods in Chapter 4. In the company of these mentor s, I also wish to acknowledge R. Francis-Floyd, who opened the door for me at The University of Florida and helped me to get started on my path. Some Chapters in this Dissertation are the resu lt of fruitful collabora tions with co-authors, who provided knowledge and resources instrumental to the work. I am indebted to D. Mann (University of South Florida), w ho provided the analytical system necessary to conduct the study in Chapter 3, along with patient teachings in th e methods of AEP audiometry and sound analysis in general. I also acknowledge his students, B. Casper, M. Hill-Cook, R. Hill, and J. Locascio, who provided guidance along the way. L. J. Gu illette and H. Hamlin (UF) taught enzyme immunoassay methods and offered their laboratory to conduct co rtisol assays included in Chapter 4. They also provided valuable advi ce in considering the stress phenomenon, advising the examination of variance among response measur es, that led to a fru itful examination of results. I also acknowledge members of their la b, in particular T. Edwards and B. Moore, for guidance. E. Adams and F. Fogarty (UF) served as undergraduate resear ch assistants along the way. Among the many (acknowledged below), thes e individuals invested tremendous effort and original thought over an extended period of time that contribute d greatly to Chapters 6 and 4 (respectively). I am very thankf ul for their sustained hard work. The Dissertation was further molded by fruitful conversations with, and wisdom contributed by, professionals in di verse fields. A. Slater (The Florida Aquarium) molded my skills in syngnathid husbandry. I owe many thanks to J. Pattee (Pioneer Hill Software) and R. Shrivastav (UF), who taught concepts of acous tics and sound analysis. A. Noxon (Acoustic

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6 Sciences Corporation) shared his knowledge in acoustic engineering th at shaped soundproofing methods that were used in the methods of Chap ters 4-6. The muting surgery that was employed in Chapters 5 and 6 was first developed and advised by D. Colson (Rhode Island ENT Physicians, Inc.), who first published a study us ing the muting method. K. Harr (UF) advised hematological methods used in Chapter 4. A. Dove (Georgia Aquarium) and E. Greiner (UF) assisted with parasite identification in Chap ter 4. L. Farina (UF) guided histological interpretation. Statistical analyses employed in th e Chapters were selected and shaped as a result of consultation with several people in addition to those previously mentioned, including M. Allen, M. Brennan, J. Colee, D. Dutterer, J. Hill, and R. Littell (UF). Several people provided technical guidance a nd offered access to technical resources that made elements of methodology possible. I am es pecially grateful to D. Petty (UF) and her laboratory (including J. Holloway and T. Crosby), who provided inva luable veterinary services all along the way to maintain the health of the research collection. They also participated in, and helped shape the methodology for, the physiological methods used in Chapter 4 and the muting surgery used in Chapters 5 and 6. D. Samu elson and P. Lewis (UF) taught histological techniques and provided access to their laboratory for histological processing and evaluation. H. Rutherford (The Pier Aquarium) loaned hydr ophones that were used extensively for sound recording. I appreciate the generous cont ributions of people in the fi shing and marine ornamental industries that provided animal s to study as well as resources to care for them. I owe many thanks to M. Helmholtz (Above The Reef), R. St evens (Mary Jane Shrimp Co.), and the crew of The Twin Rivers Marina, who believed in the project and donated many animals to build the research collection. I am gratef ul to A. and M. Maxwell and th e crew of Sea Critters, Inc., who

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7 provided a live food supply for the collection over a period of several years. R. Lewis (Aquatic Indicators) provided Mysidopsis bahia for the experiment described in Chapter 5. I enjoyed the opportunity to enga ge undergraduate students of the University of Florida in research experience. In return, this team provided a powerful work force, ensuring proper care of the research collection as well as the collection of quality data that are at the base of the results reported here. These people are E. Adams, F. Bastos, J. Bound, F. Fogarty, S. Koka, J. Liu, B. Macke, A. Maness, J. Moriarty, K. Nuessly, S. Osborn, Z. Punjani, J. Rosenbaum, B. Slossberg, D. Snipelsky, and A. Weppelman. Nine public aquaria from throughout the United States participated in The Seahorse Sound Survey (Chapter 2); for their contributions I am grateful. Thanks to R. Doege (Dallas World Aquarium), R. Curttright (Kingdom of the Seas Aquarium), K. Dobson (Maritime Aquarium), J. Reynolds and S. Reiner (Moody Gardens), S. Spina (New England Aquarium), J. Moffatt (Pittsburgh Aquarium), J. Skoy (Ripleys Aquarium of the Smokies ), J. Rawlings (Riverbanks Zoo), and K. Alford (Tennessee Aquarium). I also acknowledge N. Dunham (Florida Fish and Wildlife Conservation Commission), who graciously offered data and maps on sites in Tampa Bay where seahorses have been collected, and R. Watkins (UF), who provided Figure 2-2. Many funding sources contributed to the succes s of this Dissertation. As previously mentioned, the D.M. Smith Fellowship funded la boratory construction and research operations, and The Florida Aquarium Center for Conserva tion funded The Seahorse Sound Survey. The Mulvihill Scholarship (granted by Aquaculture Research/Environmen tal Associates, Inc. and the United States Aquaculture Association), Project A.W.A.R.E., and the Twin Rivers Marina also funded operations. The American Association of Zoo Veterinarians Mazuri Fund fully sponsored Chapter 4. My scholarship and stipen d was provided by the University of Floridas

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8 Alumni Fellowship, the Morris Animal Foundation, The Spurlino Foundation, and The Florida Aquarium Center for Conservation. Animal collection was authorized by th e Florida Fish and Wildlife Conservation Commission Special Activities License #05SR-944. Husbandry and experimental protocols were authorized by the University of Florida IACUC Protocol #D-432, the University of South Florida IACUC Protocol #2118, and The Florida Aquarium Animal Care and Use Committee.

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9 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES .........................................................................................................................13 LIST OF FIGURES .......................................................................................................................14 ABSTRACT ...................................................................................................................... .............16 CHAP TER 1 INTRODUCTION .................................................................................................................. 18 The Aquaculturists Challenge ...............................................................................................18 The Acoustic Sense: Is It Important to Consider? ................................................................. 18 Effects on Hearing ..................................................................................................................20 The Stress Response ...............................................................................................................22 The Seahorse: A Model .........................................................................................................24 Questions ................................................................................................................................27 Objectives and Outline ...........................................................................................................27 Contribution to Science and Industry ..................................................................................... 28 2 THE SEAHORSE SOUND SURVEY ................................................................................... 30 Introduction .................................................................................................................. ...........30 Materials and Methods ...........................................................................................................31 Aquarium Data Collection ...............................................................................................31 Data Collection in the Wild .............................................................................................32 Sound Analysis ................................................................................................................32 Calibration ................................................................................................................32 Sound processing ......................................................................................................33 Statistical Analysis .......................................................................................................... 34 Results .....................................................................................................................................35 Questionnaire Data ..........................................................................................................35 Tank dimensions, materials, and habitat ..................................................................35 Aeration and drainage .............................................................................................. 35 Life support equipment ............................................................................................ 36 Other noise sources .................................................................................................. 36 Animal collection .....................................................................................................36 Health and reproduction records .............................................................................. 37 Sound Analysis ................................................................................................................37 Mid-water level recordings ......................................................................................37 Bottom level recordings ........................................................................................... 38 Wild ambient noise recordings .................................................................................38

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10 Ambient Noise Comparisons ........................................................................................... 38 Effects of Design Vari ables on Total Power ................................................................... 39 Discussion .................................................................................................................... ...........40 3 AUDITORY EVOKED POTENTIAL OF T HE LINED SEAHORSE, HIPPOCAMPUS ERECTUS, AND ITS RELATIONSHIP TO CON SPECIFIC SOUND PRODUCTION ..... 54 Introduction .................................................................................................................. ...........54 Materials and Methods ...........................................................................................................56 Animal Accession, Holding, and Husbandry Procedures ...............................................56 AEP ........................................................................................................................... .......58 Experimental setup ...................................................................................................58 Sound generation, calibration, and AEP acquisition ................................................59 Data analysis ............................................................................................................60 Click Recordings .............................................................................................................60 Experimental setup ...................................................................................................60 Data analysis ............................................................................................................61 Results .....................................................................................................................................63 Ambient Noise ................................................................................................................. 63 AEP ........................................................................................................................... .......63 Click Recordings .............................................................................................................64 Pressure .................................................................................................................... 64 Particle acceleration .................................................................................................65 Comparisons between pressure wa vefor ms and particle acceleration waveforms ............................................................................................................. 65 Comparisons among individuals and between sexes ............................................... 66 Discussion .................................................................................................................... ...........66 4 SOUND, STRESS, AND SEAHORSES: THE CONSEQUENCES OF A NOISY ENVIRONMENT TO ANIMAL HEALTH ........................................................................... 82 Introduction .................................................................................................................. ...........82 Materials and Methods ...........................................................................................................83 Animal Accession and Husbandry Procedures ................................................................83 Laboratory and Experi m ental Tank Design .................................................................... 84 Sound Recording and Analysis ....................................................................................... 86 Animal Assignment and Preparation ............................................................................... 87 Ethological Methodology ................................................................................................ 87 Data collection ..........................................................................................................87 Measures and statistical analyses ............................................................................. 88 Physiological Methodology .............................................................................................89 Necropsy ................................................................................................................... 89 Blood processing ......................................................................................................90 Bacterial culture .......................................................................................................91 Measures & statistical analyses ................................................................................ 92 Results .....................................................................................................................................93

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11 Sound Analysis ................................................................................................................93 Ethological Results .......................................................................................................... 93 Ethogram .................................................................................................................. 93 State analyses ...........................................................................................................93 Event analyses ..........................................................................................................94 Physiological Results .......................................................................................................96 Morphological indices ..............................................................................................96 Hematological count ................................................................................................ 96 Leukocyte differential ..............................................................................................96 Blood glucose concentration ....................................................................................97 Plasma cortisol concentration ...................................................................................97 Incidence of disease .................................................................................................97 Discussion .................................................................................................................... ...........98 The Stress Concept ..........................................................................................................98 Primary Stress Indices .....................................................................................................99 Secondary Stress Indices ............................................................................................... 100 Tertiary Stress Indices ...................................................................................................102 Physiology .............................................................................................................. 102 Behavior ................................................................................................................. 104 Conclusions ...........................................................................................................................107 5 ACOUSTIC ROLES AND EFFECTS IN PR EY CAPT URE BEHAVIOR OF LINED SEAHORSES (HIPPOCAMPUS ERECTUS) IN AQUARIA ............................................. 118 Introduction .................................................................................................................. .........118 Materials and Methods .........................................................................................................120 Experimental Tank Design ............................................................................................ 120 Sound Recording and Analysis ..................................................................................... 120 Muting ...........................................................................................................................121 Surgical procedure ..................................................................................................121 Click recording and analysis ..................................................................................121 Prey Capture Experiment .............................................................................................. 122 Results ...................................................................................................................................123 Sound Analysis ..............................................................................................................123 Ambient noise analysis ........................................................................................... 123 Click analysis .........................................................................................................124 Prey Capture ..................................................................................................................124 Discussion .................................................................................................................... .........125 6 SOUND, SEX, AND SEAHORSES: AC OUSTI C ROLES AND EFFECTS IN COURTSHIP BEHAVIOR OF LINED SEAHORSES (HIPPOCAMPUS ERECTUS) IN AQUARIA .......................................................................................................................129 Introduction .................................................................................................................. .........129 Manifestations of Stress in Courtship Behavior ............................................................ 129 The Functions of Clicking in Courtship Behavior of the Lined Seahorse, Hippocampus erectus .................................................................................................131

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12 The Effects of Tank Noise on Acoustic Communication ..............................................131 Study Objectives ............................................................................................................132 Materials and Methods .........................................................................................................132 Animal Accession, Laboratory Desi gn, and Husbandry Procedures ............................ 132 Experimental Tank Design ............................................................................................ 133 Sound Recording and Analysis ..................................................................................... 133 Muting ...........................................................................................................................134 Courtship Experiment .................................................................................................... 134 Experimental design & ob servational m ethods ......................................................134 Ethological analysis ................................................................................................ 135 Results ...................................................................................................................................137 Sound Analysis ..............................................................................................................137 Ethological Analysis ...................................................................................................... 138 Tests of means ........................................................................................................138 Tests of mean deviations ........................................................................................139 Occurrence of clicking ........................................................................................... 141 Discussion .................................................................................................................... .........142 The Effect of Tank Noise on Courtship Behavior .........................................................142 The Functions of Clicking in Courtship Behavior ........................................................143 The Effects of Tank Noise on Acoustic Communication .............................................146 7 DISCUSSION .................................................................................................................... ...157 The Hearing Ability of Hippocampus erectus in the Context of W ild and Captive Acoustic Environments ..................................................................................................... 157 Pressure vs. Displacement .................................................................................................... 158 The Function of Clicking ......................................................................................................160 Variability in Signaling .........................................................................................................163 Masking ................................................................................................................................164 The Lateral Line ...................................................................................................................165 The Stress Response .............................................................................................................166 Physiology .................................................................................................................... .166 Behavior ...................................................................................................................... ..167 Solutions ..................................................................................................................... ..........168 REFERENCES .................................................................................................................... ........172 BIOGRAPHICAL SKETCH .......................................................................................................190

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13 LIST OF TABLES Table page 2-1 Spectra Plus processing settings ........................................................................................ 46 2-2 Public aquarium participants of the S eahorse Sound Survey ............................................ 46 2-3 Frequency distribution of tank design specifications and anim al inhabitants ...................47 2-4 Descriptive statistics of spectrum level SPLs, peak am plitude, total power, and peak frequency ..................................................................................................................... .......48 2-5 Spectrum-level SPLs at peak or dominant frequencies of representative fish sounds ...... 49 3-1 Spectra Plus recording settings. .........................................................................................72 4-1 Categorization of physiological m easures for statistical analysis .................................... 109 4-2 Ethogram of the lined seahorse, Hippocampus erectus: Maintenance behavior ............ 110 4-3 Summary of physiological results ....................................................................................111 5-1 Partial ethogram of the lined seahorse, Hippocampus erectus: Prey capture behavior .. 127 6-1 Partial ethogram of courtship behaviors of the lined seahorse, Hippocampus erectus ...148 6-2 Summary of significant results of Type III tes ts of means of fixed effects in SAS PROC GLIMMIX ............................................................................................................ 149 6-3 Summary of significant results of Type III te s ts of mean deviations of tank treatment effects or its interactions in SAS PROC GLIMMIX ....................................................... 150

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14 LIST OF FIGURES Figure page 2-1 The Seahorse Sound Survey kit .........................................................................................50 2-2 Acoustic sampling sites in Tampa Bay, FL. ......................................................................51 2-3 Power spectra from representative tanks ........................................................................... 52 2-4 Total power vs. selected design specifications ..................................................................53 2-5 Mid-level total power vs. wall m aterial type among ta nks with gravel bottoms ............... 53 3-1 Soundproofed laboratory tank ............................................................................................73 3-2 AEP experimental setup .....................................................................................................74 3-3 Auditory evoked potentials to a 400 Hz tone pip .............................................................. 75 3-4 Click recording chamber ................................................................................................... .76 3-5 Click characteristics measured in the tim e domain waveform .......................................... 76 3-6 Click characteristics measured in the frequency dom ain waveform ................................. 77 3-7 Representative power spectra of sound pr essure of long-term holding tanks and the sound-proofed laboratory tank ...........................................................................................78 3-8 Power spectrum of particle accelerat ion of the sound-proofed laboratory tank. ............... 79 3-9 Audiograms of the lined seahorse, H. erectus, for sound pressure and particle acceleration .................................................................................................................. ......79 3-10 A resonant click, depicted in the time domain ................................................................... 80 3-11 Sound pressure audiograms of representative hearing generalist fishes, m easured by the AEP technique ..............................................................................................................80 3-12 Comparison of the broadband sound pressu re audiogram against peaks of recorded clicks ........................................................................................................................ ..........81 3-13 Comparison of the broadband particle acceleratio n audiogram against peaks of recorded clicks ...................................................................................................................81 4-1 Experimental tank design schem atic: Loud tank ............................................................ 112 4-2 Experimental tank design schem atic: Quiet tank ............................................................ 113

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15 4-3 Power spectra of ambient noi se in representative tanks .................................................. 114 4-4 Proportion of time spent stationary .................................................................................. 115 4-5 Number of adjustments made per hour while stationary ................................................. 115 4-6 Occurrence of piping ...................................................................................................... ..116 4-7 Occurrence of clicking .................................................................................................... .116 4-8 Occurrence of gaping ...................................................................................................... .117 5-1 Power spectra of ambient noi se in representative tanks .................................................. 128 6-1 Power spectra of ambient noi se in representative tanks .................................................. 151 6-2 Comparisons of means (+ SE) of male approach ............................................................152 6-3 Comparisons of means (+ SE) of female point ................................................................152 6-4 Comparisons of means (+ SE) of clicking between males and females .......................... 153 6-5 Comparisons of standard deviations of brightening between ma les in quiet and loud tanks ......................................................................................................................... ........153 6-6 Comparisons of standard deviations of di splay between m ales in quiet and loud tanks 154 6-7 Comparisons of standard devi ations of display among fe males ......................................154 6-8 Comparisons of standard deviations of pointing between m ales in loud and quiet tanks ......................................................................................................................... ........155 6-9 Comparisons of standard deviations of pouch pumping between m ales in loud and quiet tanks ................................................................................................................... .....155 6-10 Comparisons of standard devi ations of pointing among fe males .................................... 156 6-11 Comparisons of standard devi ations of clicking among m ales ........................................ 156 7-1 Comparisons of ambient noise in tank environm ents with broadband hearing in Hippocampus erectus .......................................................................................................170 7-2 Comparisons of ambient noise in the wild with broadband hearing in Hippocampus erectus ..............................................................................................................................171

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16 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE FUNCTIONS OF SOUND PRODUCT ION IN THE LINED SEAHORSE, HIPPOCAMPUS ERECTUS, AND EFFECTS OF LOUD AM BIENT NOISE ON ITS BEHAVIOR AND PHYSIOLOGY IN CAPTIVE ENVIRONMENTS By Paul August Anderson August 2009 Chair: William J. Lindberg Major: Fisheries a nd Aquatic Sciences Loud noise in aquaria represents a cacophonous environment for captive fishes. I tested the effects of loud noise on acoustic communication, feeding beha vior, courtship behavior, and the stress response of the lined seahorse, Hippocampus erectus. Total Root Mean Square (RMS) power of ambient noise to which seahorses are exposed in captivity varies widely but averages 126.1 + 0.8 deciBels with reference to one micropas cal (dB re: 1 Pa) at the middle of the water column and 133.7 + 1.1 dB at the tank bottom, whereas am bient noise in the wild averages 119.6 + 3.5 dB. Hearing sensitivity of H. erectus, measured from auditory evoked potentials, demonstrated maximum spectrumlevel sensitivities of 105.0 + 1.5 dB and 3.5 X 10-3 + 7.6 X 10-4 m/s2 at 200 Hz; which is characteris tic of hearing generalists. H. erectus produces acoustic clicks with mean peak spectrum-level amplitudes of 94.3 + 0.9 dB at 232 + 16 Hz and 1.5 X 10-3 + 1.9 X 10-4 m/s2 at 265 + 22 Hz. Frequency matching of clicks to best hearing sensitivity, and estimates of audition of broadband signals suggest that seahorses may hear conspecific clicks, especially in terms of particle motion. Behavioral investigations revealed that clicking did not improve prey capture proficiency. However, animals clicked more often as time progressed in a courtship sequence, and mates

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17 performed more courtship behaviors with cont rol animals than with muted animals, lending additional evidence to the role of clicking as an acoustic signal during courtship. Despite loud noise and the role of clicki ng in communication, masking of the acoustic signal was not demonstrated. Seahorses exposed to loud noise in aquaria for one month demonstrated physiological, chronic stress responses: reduced weight and body condition, and in creased heterophil to lymphocyte ratio. Behavioral alterations were characterized by greater mean and variance of activity among animals housed in loud tanks in the first week, followed by habituation. By week four, animals in loud tanks demonstrated variable performance of clicking and piping, putative distress behaviors. Despite the physiological stress response, animals in loud tanks did not reduce feeding response or courtship behavior, suggesting allostasis.

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18 CHAPTER 1 INTRODUCTION The Aquaculturists Challenge In general, the aim of finfish aquaculture is to maximize production of healthy finfish for economic gain. Biologically, this translates into maximizing gr owth, growth rate, rate of reproduction, and fecundity of target organisms. The culturist mu st give consideration to the many factors needed to ensure survival and reproduction of his fish. This includes considering the animals nutritional needs that vary over the animals life history (Watanabe, 1988), designing and maintaining a filtration system a nd husbandry schedule that keeps water quality within acceptable parameters (Bromage et al., 1988), giving consideratio n to stocking density and tank size (Pillay, 1990), and providing an en vironment with necessary environmental cues sufficient to induce spawning (Bardach a nd Magnuson, 1980), among other considerations. The strategies that an aquaculturist may us e to improve one parameter might adversely affect another. For example, while a high rate of water flow might facilitate filtration, the increased current may adversely aff ect delicate fry (e.g., Opstad et al., 1998). A feed that is high in protein may accelerate growth but also may in crease biological load on the filtration system and adversely affect water quality ( e.g., Tidwell et al., 1996). Some how, the aquaculturist must balance numerous factors to produce an optimal environment for his organism. This can be particularly challenging when some strategies may adversely affect an organism in ways that the aquaculturist is unaware. The Acoustic Sense: Is It Important to Consider? I suggest that the acoustic sense of fish is a sen sory modality that is often overlooked in aquaculture. The earliest evolved and most general role of the fish ear is to gain information about the environment through its acoustic signature (Popper and Fa y, 1999). Some fishes have

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19 taken further advantage of the acoustic sens e by evolving sound production mechanisms for intraspecific communication, as in the plainfin midshipman fish (Porichthys notatus) that utilizes acoustic communication in courtship (Bass and Clark, 2003). An intensive culture system may significantly interfer e with both functions. Fish in intensive culture systems are exposed to ambient noise from many sources, perh aps most notably the pump motors that run to maintain water quality. Sounds from water pum ps, air bubbles, air pumps, chiller motors, and the like can combine to create a loud, cacophono us sound in a tank across a broad frequency range (Bart et al., 2001; Davidson et al., 2007). What effect this has on fish health, growth, courtship, and reproduction in the co ntext of aquaculture has been explored only cursorily in the literature. Banner and Hyatt (1973) expos ed eggs and larvae of Cyprinidon variegatus and Fundulus similis to noise (at 118 peak deci bels with reference to one micropascal, or dBpeak re: 1 Pa) from a submersible water pump and airstones. Tested against controls in quiet tanks (at 103 dBpeak re: 1 Pa), they discovered greater mortality of eggs and fry of C. variegatus in noisy tanks, and slower growth rates of fry in noisy tanks in both sp ecies. Lagardre (1982) exposed brown shrimp ( Crangon crangon ) to noise (at 128 dBpeak re: 1 Pa) by aquarium air pumps placed adjacent to culture tanks. Tested against controls in quiet tanks (at 88 dBpeak re: 1 Pa), animals in noisy tanks demonstrated slower growth, less food consumption, reduced reproduction (less egg-bearing females), and higher mortality due to higher rates of cannibalism and higher incidence of disease. In a la ter study, Regnault and Lagardre (1983) also documented increased oxygen consumption and ammonia excretion (suggesting increased metabolism) among brown shrimp in loud tanks. In contrast, Wysocki et al. (2007) found no

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20 effect of chronic exposure to loud tank noise (at up to 150 dBrms re: 1 Pa) on growth or mortality of rainbow trout (Oncorhynchus mykiss). Outside the context of a quaculture, it is clear that sound can affect fish at all levels of biological organization, ranging from biochemical perturbations ( e.g., Santulli et al., 1999), to physiological changes ( e.g., Sverdrup et al., 1994), to behavioral modifications ( e.g., Pearson et al., 1992). Most of this work has examined the e ffects of either laborato ry-produced sounds, that are unlikely to be heard in a natural or culture environment, or anthropogenic sounds encountered in nature. Effects on Hearing Som e of the earliest work has studied the effect s of noise on the auditory system of fish. Popper and Clarke (1976) used classic behavi oral training techniques (avoidance conditioning, threshold tracking, and classical conditioning of re spiratory suppression) to show that goldfish (Carassius auratus) exposed to intense tonal stimulation in creased hearing thresholds; threshold shifts varied with different frequencies of exposure and testing. Also using classical conditioning techniques, Fay (1974) measured threshold increases of tones masked by broadband noise. More recently, workers have applied the a uditory evoked potential (AEP) technique to measure fish hearing. The AEP technique is a non-invasive far-field r ecording of synchronous neural activity in the eighth ne rve and brainstem auditory nuclei elicited by acoustic stimuli (Jacobson, 1985). Originally invented and devel oped for use in clinical evaluations of human hearing (Jacobson, 1985), the AEP technique has sin ce been applied to many animals, including mammals ( e.g., Wolski et al., 2003) and fish ( e.g., Yan, 2001). Scholik and Yan (2001a&b, 2002a&b) took advantage of the AE P technique to study the effect s of noise on the auditory sensitivity of the fathead minnow ( Pimephales promelas), a hearing specialist (a fish that has

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21 evolved a morphological specialization to increas e hearing sensitivity, such as a bony or gaseous vesicular connection between the gas bladder and the inner ear), a nd the bluegill sunfish ( Lepomis macrochirus ), a hearing generalist (without such a morphological specialization). Using this technique, they exposed both fish to white noise (a broadband noise in which all frequencies in the noise spectru m are of the same sound pressure level) between 0.3 and 4.0kHz at 142 dB re: 1 Pa for the minnow and 0.3 to 2.0 kHz at 142 dB re: 1 Pa for the sunfish for various durations. In addition, they exposed minnows to two hours of boat engine noise, at a sound pressure level of 142 dB re: 1 Pa. In noise-exposed minnows, auditory thresholds were significantly elevated at several frequencies test ed within the minnows most sensitive hearing range. The auditory effect of noise exposur e was dependent on duration of exposure. In contrast, the sunfish demonstrated a slight, but insignifi cant, threshold increas e. They concluded that ambient noise in natural environments could have negative impacts on fish, but that effects may differ between fish base d on hearing sensitivity. Fishes may still fail to detect biologically relevant sounds even if a temporary hearing threshold shift (i.e., hearing loss) does not occur. Si gnals may simply be masked by the presence of loud ambient noise. The masking e ffect in fishes has been recognized since 1961 (Tavolga, 1967). Among hearing specialist fish es, masking can be quite pronounced at very low levels of noise. In goldfish, Fay (1974) documented an increase in signal detection threshold of approximately 20 dB when a spectrum level maski ng noise of only 51 dB re: 1 Pa was present. Masking also occurs in hearing generalist fishes, though the relativ ely poor hearing sensitivity of generalists requires that maski ng noise be substantially louder to produce an effect. A few studies demonstrated masking among representative heari ng generalists ( Lagodon rhomboides, Caldwell and Caldwell, 1967, in Tavolga, 1974; Tilapia macrocephala, Haemulon sciurus,

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22 Tavolga, 1974; Gadus morhua, Hawkins and Chapman, 1975; Sebastes schlegeli, Motomatsu et al., 1998; Lepomis gibbosus, Wysocki and Ladich, 2005; Perca fluviatilis, Amoser and Ladich, 2005) at critical ratios (t he difference between hearing thres hold and spectrum level of masking noise) ranging from 10 to 60 dB (median around 20 dB), with spectrum level masking noise ranging from 70 to 90 dB re: 1 Pa and total power of masking noise ranging from 100 to 110 dB re: 1 Pa (where measured). Furtherm ore, broadband noises cause a more pronounced masking effect than do narrowband noises (Haw kins and Chapman, 1975), thus the broadband nature of ambient noise is of additional concern. Masking may pose substantial problems for an imals that rely on acoustic communication. Many fish produce sounds during aggression, defense, territorial advertisement, courtship, and mating (for an overview, see Zelick et al., 1999). Masking of these signals in aquaria may result in, for example, excessive and unnecessary aggressi ve encounters (and subs equent social stress) if acoustic territorial si gnals are not perceived ( e.g., Myrberg and Riggio, 1985), inhibition of species recognition ( e.g., Spanier, 1979), or inhibition of phonotaxis, courtship, and/or mating behavior ( e.g., Stout, 1975; McKibben and Bass, 1998). The Stress Response Alternatively, am bient noise should be consid ered as a possible stressor in a culture system. Stress is an important consideration in the successful husbandry of finfish. Severe stress can result in mortality, but even sublethal st ress can compromise various physiological and behavioral functions, leading to suppressed dise ase resistance, growth rate, and fecundity, all contributing to suboptimal pr oduction (Iwama et al., 1997). The stress response is an evolutionarily adapted cascade of physiol ogical and behavioral changes that occur to enable the animal to react adaptively to a stressful stimulus, or to cope long-term in a stressful environmen t (the latter scenario has been coined allostasis, essentially

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23 an alternative to homeostasis in a suboptimal environment, by Sterling and Eyer, 1988). Ultimately, if the persistence of a chronic stresso r overcomes the ability of an animal to cope, pathological conditions and mort ality can ensue (per the General Adaptation Syndrome, or GAS, Selye, 1950). The adaptive components of the stress response are mediat ed endocrinologically via the secretion of catecholamines and cortisol, often considered as indices of a primary stress response (Sumpter, 1997). Together, these horm ones cause a suite of changes in the physiology of the animal, considered as secondary stress indices. These include (but are not limited to) changes in the matrix of ionic, osmotic, and acid-base components of the blood (McDonald and Milligan, 1997), immune system depression (Balm, 1997), downregulation of reproductive hormones, and variable changes in growth horm one plasma concentrations (Pankhurst and Van Der Kraak, 1997). Tertiary, or w hole-organism indices, include m easures such as behavioral changes (Schreck et al., 1997), and depression in growth, weight ga in, fecundity, and survival of offspring (Pankhurst and Van Der Kraak, 1997). While acoustic stress has been suggested as a possible factor affecting aquaculture ( e.g., Lagardre, 1982; Regnault and Lagardre, 1983; Wysocki et al., 2007), other literature has supported anthropogenic noise as a cause of biochemical, physiologi cal, and behavioral changes, all markers of stress response ( e.g., Blaxter and Batty, 1987; Knudsen et al., 1992; Skalski et al., 1992; Sverdrup et al., 1994; Knudsen et al., 1997; Santulli et al., 1999). Santulli et al. (1999) and Sver drup et al. (1994) examined th e effects of intense acoustic stimulation emitted by air guns used in seismic surveys on biochemical and physiological responses in European Sea Bass (Dicentrarchus labrax L. ) and Atlantic Salmon (Salmo salar) respectively. Santulli et al.s (1999) post-shock serum analyses i ndicated increases in cortisol, variations in glucose and lactat e, and decreased adenylate concentrations. Skeletal muscle and

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24 liver ATP concentration fell, ADP rose, while AMP did not significantly ch ange. Sverdrup et al. (1994) demonstrated an immediate post-shock decrease in cortisol followed by a consistent rise to 40 nmol/L over pre-shock levels. Adrenaline levels spiked at 75 nmol/L over pre-shock levels. Structurally, the vascul ar endothelium of the ventral ao rta and the coeliaco mesenteric artery (CMA) revealed signs of injury within th e first 30 minutes after the experimental shock. Functionally, the cholinergic and adrenergic vasoconstrictor responses in the CMA were markedly reduced during the first day after the shock. The loss of structural integrity and the reduced functional responses indicated a temporar y impairment of the vascular endothelium in response to the seismic shock. Behavioral responses to sound stimulation have also been examined. Blaxter and Batty (1987) demonstrated startl e responses in herring (Clupea harengus harengus) when exposed to transient sound stimuli. Knudsen et al. (1992, 1997) demonstrated spontaneous avoidance responses of Atlantic and Pacific salmon ( Salmo salar and Oncorhynchus tshawytscha, respectively) in response to 10 Hz stimulation, hypothesizing acute awareness in the infrasound range to the evolutionary impor tance of detecting swimming pred ators. Skalski et al. (1992) demonstrated alarm and startle responses of rockfish (Sebastes spp.) to acoustic stimuli emitted by geophysical survey devices, and tied these re sults to a decreased catch-per-unit effort. The Seahorse: A Model Seahorses ( Hippocampus spp.) have received much attentio n in recent years in several fields of study. Classical biol ogists have taken advantage of the genus unique, monogamous mating system to test predictions involving sexu al selection and sex ro les (Vincent et al., 1992; Vincent, 1994a&b; Masonjones and Lewis, 2000) while exploitation of seahorses in the Traditional Chinese Medicine and aquarium market s (Lourie et al., 1999) ha s fueled interest in

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25 seahorse aquaculture as a means to provide alternativ e sources to wild-caught animals (Lockyear et al., 1997; Woods, 2000; Job et al., 2002; W oods, 2003). Several detailed studies on sea horse courtship and mating beha vior suggest that seahorses use visual cues in mate choice and timing of ma ting. Seahorses pair size-assortively (Vincent and Sadler, 1995; Jones et al., 2003 ), and may engage in ritua listic courtship behaviors to synchronize reproductive states between pa ired individuals (Vincent, 1995). However, the role of sound communication in seahorse pairing or courtship has remained largely unstudied. Dufoss (1874, cited in Fish, 1953) first repor ted a monotonous noise analogous to that of a tam bour, especially during the breeding season in courting Hippocampus brevirostris Fish (1953) also provided two other accounts of seahorses (species unknown) making clicking noises when placed in proximity to one another in separate jars, suggesting an intraspecific signaling system. She first characterized the clicking noise made by H. hudsonius (= erectus) when placed in a new aquarium environment. Her recordings indicated broadband signals ranging from 0 to 4.8 kHz, with maximum energy between 300 to 600 Hz and 400 to 800 Hz. She hypothesized that sound production may be used in new surroundings for orientation, and perhaps to find conspecifics. Fish (1954, cited in Marshall, 1966) later observed clicking in courtship and mating. More recently, Vincent (1994b) observed H. fuscus snapping in the context of male-male competition durin g courtship with a female, while Woods (2000) observed clicking by H. abdominalis while rising in the water column, prior to (or perhaps during) egg transfer. But clic king is not included among courtshi p behaviors described between mating pairs of H. fuscus (Vincent, 1994b), H. whitei (Vincent and Sadler, 1995), or H. zosterae (Masonjones and Lewis, 1996).

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26 Colson et al. (1998) conducted a detailed study of sound producti on during feeding in H. zosterae and H. erectus They characterized a similar cl icking sound, stridulatory in origin, produced by an articulation between the ridge on th e dorsal posterior region of the supraoccipital and the medial groove on the anterior margin of the coronet. Peak frequencies of clicks produced by H. erectus ranged from 1.96 to 2.37 kHz. Furt hermore, they found that peak frequencies of clicks were inversely correlated with body weight, at least in H. zosterae The higher range of peak freque ncies (2.65 to 3.43 kHz) for H. zosterae a much smaller species at 3 vs. 15 cm maximum height (Kuiter, 2000), further supports this inverse relationship. While this clicking-sound was associated w ith feeding, the authors also ob served this sound and related behavior (a rapid, upward jerk of the head) when seahorses were placed into a new aquarium, and among competing males during courtship. From the perspectives of conservation and aquaculture, wild seahorse populations are declining (Project Seahorse, 2009), promp ting collection regulations (CITES, 2001). Stakeholders are thus explor ing aquaculture as an alternative to wild collection ( e.g., Wilson and Vincent, 1998). However, seahorses have proven pa rticularly challenging to culture. Matsunaga and Rahman (1998) provided evidence suggestin g that seahorses do not have gut-associated lymphoid tissue (GALT), an important component of adaptive immunity. Seahorses are also particularly vulnerable to di sease in aquaculture conditions (Vincent, 1998, Berzins and Greenwell, 2001). Stressors su ch as crowding, handling ( e.g., Barnett and Pankhurst, 1998), and ambient noise (e.g., Lagardre, 1982) might further comp romise the already immunologically disadvantaged seahorse, potential ly rendering it vulnerable to pathogens commonly encountered in aquaculture conditions (e.g., Leonard et al., 2000).

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27 The seahorse is thus an opportune model to measure the effects of ambient noise on the health, welfare, and behavior of fish in an aquacu lture setting. The vulnerabi lity of wild seahorse populations urges research and development of aquaculture techniques to improve production, in the hopes of alleviating fishing pressure. Their documented and stereotypi cal courtship behavior facilitates behavioral measurements of courts hip and reproduction and the effects that noise stress may have on them. Their unusual immune system may predispose them to stress-induced disease, prompting the evaluation of stress resp onse to chronic noise exposure. Seahorses are known to produce sounds, but the function of sound production is st ill unclear in this group of animals. This presents an opportunity to test sound production in the context of feeding (prey capture) and courtship, among othe r behaviors, and, as it relates to ambient noise, potential masking effects of ambient noise on sound production and communication. Questions Does am bient noise impact fish health, behavior, and physiology in aquaculture settings? If so, are effects mediated via a stress response that may be manifested in measures of health, behavior, or physiology? Or, doe s ambient noise mask acoustic signals that may be important components of behavior in fishes? Objectives and Outline The objectives of this dissertati on are severalfold. In Chapter 2, I seek to c haracterize the range of ambient noise to which captive seahorses are exposed in aquaria, to ascertain the effect of tank design elements on ambient noise, and to explore potential correlations between ambient noise and seahorse morbidity or mortality. In Chapter 3, I seek to 1) characterize the hearing range of the lined seahorse, H. erectus, 2) characterize the acoustic properties of the seahorse click, and 3) assess audition of conspecific sound production. In Chapter 4, I explore the effects of chronic loud noise exposure on physiologi cal and behavioral measures of stress in H. erectus.

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28 In Chapters 5 and 6, I assess th e functions of sound production (clicking) on prey capture and courtship behavior, respectively, the effect of chronic loud nois e exposure on these behaviors, and explore alternative hypothese s explaining the mechanism of a ny observed effect: 1) that ambient noise may contribute to reductions in th ese behaviors via stress response, or 2) that ambient noise may mask acoustic signaling, di srupting the acoustic component of these behaviors. Contribution to Science and Industry This dissertation addresses quest ions that are im portant and in teresting to the fields of ethology and aquaculture. Behaviorally, this proposal addresses an important umwelt question. To understand the biological systems of an animal, it is important to study how an animal perceives its environment. Failing to do so could mean the failure to detect information that an animal is processing to make adaptive deci sions. This shortcoming can hamper the understanding of signaling and co mmunication systems, sexual sel ection, foraging, prey capture, and orientation (Wersinger and Martin, 2009). Failing to understand an animals umwelt may have important consequences in application/industry. The field of aquaculture relies on anim als to reproduce naturally (or sometimes induced with hormones). While e fforts are made to provide an environment conducive to spawning, some operators may be ove rlooking the acoustic environment. Breeding tanks may be exposed to sound and vibration from heavy circulation and filtration equipment. The underwater cacophony that results may either mask acoustic signals that are important components of behaviors such as courtship and pr ey capture, or may be stressing fish, resulting in reduced health, grow th, and reproduction. Results of this dissertation may prompt aqu aculturists to consider the acoustic environment of their systems, and to reduce ambient noise where possible via system design modifications.

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29 The results presented here may be useful not only to the stakeholders located across the globe who have recently begun culturing seahorses to provide an altern ative source for product in the traditional Chinese medicine and aquarium markets, but also to culturists of all types of aquatic animals found worldwide.

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30 CHAPTER 2 THE SEAHORSE SOUND SURVEY Introduction Given the ef fects of chronic noise exposure on fish hearing, behavior and physiology, it is important to understand whether or not ambient noi se in aquarium/aquaculture conditions is loud enough to induce such deleterious effects. However, ambient noise levels commonly encountered in aquarium and aquaculture se ttings remain largely uncharacterized. Bart et al. (2001) surveyed ambient noise in several different aquaculture settings; indoor circular fiberglass and concrete tanks, indoor concrete and wooden-frame raceways, and outdoor ponds. Of all enclosure types, ponds without runni ng aerators were the quietest at a total power of 94 dB re: 1 Pa, but pond aerators increased to tal power to 135 dB. In a frequency range of 25-1000 Hz, concrete tanks were typically more qui et (at 110 dB SPL re: 1 Pa total power) than fiberglass tanks (at 130 dB). Some of these SPLs clearly fa ll into a range known to induce hearing loss and stress, though physical inne r ear damage occurs at higher SPLs ( e.g., 180 dB SPL re: 1 Pa, Hastings et al., 1996; McCauley et al., 2003). Here, I characterize the range of am bient noise to which seahorses ( Hippocampus spp., Family Syngnathidae) are exposed in public aquaria. This survey offers data on an assortment of smaller tanks more likely to be in use among aqua rists and culturists of ornamental fishes. It includes summarized data on the variety of tank sizes, materials used for tanks and stands, as well as drainage, aeration, and associated noi se-producing life support equipment. Animal collections, health, and reproduction records ar e summarized. Ambient noise profiles are characterized, and relationships between tank desi gn specifications and ambient noise levels are explored and presented. Finally, I present ambient noise data collected from geographic areas in

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31 Tampa Bay, FL (USA) within which wild populations of H. erectus occur, to provide a comparison between noise encountered in the w ild and noise encountered in captivity. Materials and Methods Aquarium Data Collection The Seahorse Sound Survey was a kit deve loped for participation by public aquaria throughout the United States. The kit containe d a questionnaire, an HTI-96m in pressuresensitive hydrophone (High Tech Instruments, Inc., sensitivity: -165 dB re: 1 V/Pa, bandwidth: 2-30,000 Hz), a Creative NOMAD J ukebox 3 digital audio recording device, a digital camera, an instruction manual, hea dphones and measuring tape (Figure 2-1). The questionnaire requested the following data: Contact Information Tank Specifications: Volume, dimensions, tank wall and stand materials, substrate type Aeration: Air blower vs. pu mp, type, number of air endpo ints, type of air endpoint Drainage: Number, location Heaters/Chillers: Types, distance from tank, method of plumbing/attachment Pumps: Types, distance from ta nk, method of plumbing/attachment Other Noise Sources: Types, distance from tank, method of plumbing/attachment Animal Collection: Species, number of males, females, juveniles, unknown sex, and breeding pairs Breeding Records: Number of broods per pair per year, number of fry Health & Disease Records: Type and number of sick and/or dead animals per year In addition to completing the questionnaire, participating aquarists were asked to take two 1 min recordings with the hydrophone in two positi ons: 1) in the middle of the water column, and 2) touching the tank bottom. During reco rding, the hydrophone was c onnected to the 9-volt

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32 battery amplifier, and to the NOMAD Jukebox 3. Aquarists were asked to hold the NOMAD Jukebox 3 and excess cord still du ring recording, and not to allo w recording equipment to come in contact with any other solid during recording. Recordings were typically taken with a 0 dB gain, but gain was adjusted (+ up to 12 dB) if ambient noise was too quiet or too loud at 0 dB gain, and corrected in calibration for subsequent analysis. Record ings were digitized as .wav files with a sampling rate of 44.1 kbps. Finally, photos were taken of tanks and systems to corroborate questionnaire data. Data Collection in the Wild On July 15, 2008, acoustic recordings were ta ken from ten sites within and around Tampa Bay, FL (USA) at GPS locations where trawls ha d previously collected 3 or more seahorses, between the years of 2006 and 2007 (N. Dunham, Florida Fish and Wildlife Conservation Commission, pers. comm., Figure 22). Acoustic recording equipm ent consisted of an HTI-96 min hydrophone (specs as above) with a 17 m cable and the Creative NOMAD Jukebox 3 digital audio recording device. At each recording site Beaufort number (Wenz, 1962) and depth were recorded for characterization of environmental condition and site respectively. The hydrophone was lowered overboard until resting on the seafloor, and 1-min acoustic recordings were made with the Jukebox set at 0 dB Gain. The boat engi ne was shut off during r ecordings, and the cable was held still to prevent ac oustic artifacts due to jostli ng of the cable/hydrophone. Sound Analysis Calibration All reco rdings were analyzed with SpectraPl us (Pioneer Hill Software) signal analysis software. For calibration, I generated a 500 Hz sine wave at 1 Vpeak with a Leader LG1301 2 MHz function generator connected to a Lead er LS1020 20 MHz oscilloscope for signal visualization. The function generator wa s also connected to the NOMAD Jukebox 3, and

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33 recordings of the signal were made at all gain levels, and digitized as .wav files with a sampling rate of 44.1 kbps. Calibration setting files in SpectraPlus we re created using these knownvoltage signals, according to pr ogram instructions, for all gain levels of the NOMAD Jukebox 3. Sound processing Sound files were post-processed by rem oving putative artificial electrical peaks in frequency spectra (at 60 Hz or its harmonics) usi ng the Fast Fourier Transf orm (FFT) notch filter function in CoolEdit (Syntrillium Software Corpor ation). These notch-filtered files were then post-processed in SpectraPlus using the analysis settings summarized in Table 2-1. I chose a decimation ratio1 of 22 along with an FFT-size of 2048 to achieve a spectral line resolution of approximately 1 Hz (0.979 Hz) and an upper frequency detection limit of 1,002 Hz. These settings enable accurate spectrum level SPL meas urements and output total power SPLs that are summed over a frequency range of 2-1,002 Hz. Hear ing generalist fishes, without connections between the gas bladder and inne r ear, generally have insensi tive hearing above 1,000 Hz (Fay, 1988b). Hearing tests of H. erectus using the auditory evoked pot ential (AEP) technique are consistent with this observation (Chapter 3). In addition, there are no observable connecting structures between the swimbladder and the inner ear in this species (pers. obs.). Both lines of evidence thus suggest that seahorses, or at least H. erectus, are/is (a) hearing generalist(s). In their survey of aquaculture syst ems, Bart et al. (2001) also an alyzed recordings in the low frequency region (25-1000 Hz), th at provides an equiva lent frame of reference for comparison. It should be noted that a low pass filter reduces the total power, but the resulting total power SPL is summed only over the fishs range of envi ronmentally relevant hearing ability. Unless 1 Decimation reduces the sampling rate of the signal by averaging (in this case) every 22 samples as one sample. In combination with an FFT-size of 2048, this procedure impr oves the frequency resolution of the power spectrum to a 1 Hz bandwidth.

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34 otherwise indicated, all am plitudes reported in the results are peak amplitudes in deciBels with reference to 1 micropascal (dB SPL re: 1 Pa). Statistical Analysis Questionnaire data were tabulated and descrip tive statistics w ere computed. Power spectra of all ambient noise recordings were generated; from these, I obt ained spectrum level SPLs at 10, 20, 30, 40, 50, 70, 80, 90, 100, 200, 400, 500, 700, 800, and 980 Hz, and recorded peak frequencies, peak amplitudes, and total power SPLs. These data were tabulated and descriptive statistics computed. I conducted a paired t test between total power SPLs at the middle of the water column and at the tank bottom, and unpaired t tests between total power SPLs of wild ambient noise recordings and tank recordings at both positions. To examine possible trends between tank/filtration design variables and ambien t noise, or animal bree ding/health records and ambient noise, I constructed scatte rplots setting total power SPLs on the y-axis. I fit regression lines to plots with continuous x-variables. For ordinal or categorical xvariables, plots were visualized for trends. I also conducted unpaired t tests for mid-level and bottom-level total power SPLs between tanks containing adult males and female seahorses th at produced at least one brood and tanks that did not produce broods. Only stand material ty pe, tank wall material type, and substrate type revealed possible visual trends among all constructed plots. These three variables were tested in a th ree-way MANOVA using SAS software according to the following model: y1 + y2 = w + s + b + w*s + w*b + s*b + w*s*b + (2-1) where: y1 = mid-level total power (dB SPL re: 1 Pa), y2 = bottom-level total power, w = wall material type, s = stand material type, b = bottom habitat type, and = experimental error. All three independent variables are class variables. Data were not balanced, so Type III sums of squares were used. For MANOVA results, the W ilks Lambda statistic was used to assess

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35 significance. Within component ANOVAs, Tukeys test comparisons were made among treatment means of significant de sign variables. Significant inte raction effects were further explored using PROC SORT to test simple effect s of one factor at fixed levels of the other, followed with Tukeys test comparis ons among sorted treatment means. Results Questionnaire Data Nine public aquaria returned data from 42 tanks (Table 2-2). Results of questionnaire data described below m ay be cross-referenced with Table 2-3. Tank dimensions, materials, and habitat Tank sizes ranged from 19 to 10,978 L, with a median of 291 L. Most tanks were rectangular ( n = 31), others were cylindrical, hexagona l, other polygonal, crescent, bubble or bow-shaped. Tank walls were constructed of fiberglass ( n = 14), glass ( n = 13), acrylic (n = 11), or concrete ( n = 3). Stands on which tanks were s upported were constructed of plastic ( n = 15), wood ( n = 13), metal (n = 9), or concrete ( n = 4). The bottom habitat of tanks were primarily gravel (n = 26), but some tanks had bare bottoms ( n = 9), while others had a plenum underneath gravel beds ( n = 6). Aeration and drainage Twenty-two tanks were aerate d. Seventeen tanks were aerated by a rem ote blower, three by air pump (2 tanks had unreported sources). Of aerated tanks, 13 had one air endpoint, 8 had two air endpoints, and one had f our endpoints. Twelve of the ae rated tanks had open end airlines in the tank, and ten had airstones. Most tanks had one drain ( n = 29), six had two drains, one tank had three drains, and five tanks had no drai nage. Of the tanks with drainage, most (n = 32) were surface skimmers, and just four were subsurface drains.

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36 Life support equipment Twenty-nine tanks were serviced by heaters or heat exchang e units that were either plumbed inline ( n = 12) at 0.6 + 0.3 m (mean + SE) from the tank, inst alled in the sump ( n = 11), or in the tank itself ( n = 6). Only four tanks were serviced by chillers, either plumbed inline or located in the sump, at a median of 3 m from th e tank (range: 0.5 to 7.6 m) Eighteen tanks were serviced by one water pump, nineteen were serv iced by two water pumps, and two by three water pumps (or had multiple water pumps servicing one system with multiple tanks). Wattage of primary pumps ranged from 20 to 1492 W, with a median of 265 W. Thirty-five primary pumps were plumbed inline using PVC, one plumbed w ith vinyl tubing, one submersed in the sump, and two submersed in the tank. Primary pumps were located up to 7.6 m away from tanks, with a median distance of 0.9 m. Wattage of sec ondary pumps ranged from 25 to 746 W, with a median of 322 W. Fourteen were plumbed inline using PVC, six were plumbed inline with vinyl tubing, and one was submersed in the tank. Secondary pumps had the same range and median distance as primary pumps. Tertiary pumps for two tanks were 218.5 W pumps plumbed in-line with PVC and located 0.3 m from the tank. Other noise sources Other noise sources not associated w ith ta nks included pumps, chillers, television and audio, and drainage overflows. These noise sources were located between 0.3 to 3 m from tanks, with a median distance of 1.5 m. Animal collection Hippocampus species kept included H. abdominalis, H. erectus, H. fuscus, H. kuda, H. procerus, H. reidi, H. subelongatus, and H. zosterae. Only five tanks kept m ore than one Hippocampus species. Tanks held anywhere from zero to 80 individuals, w ith a mean of 8.5. Tanks containing adults were stoc ked to a mean density of 19 + 3 adults/m2. Average sex ratio

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37 (M:F) of animals of known sex was 0.97. Ten ta nks housed between 2 and 54 juveniles, with a median of 7. Tanks containing juveniles were stocked to a mean density of 38 + 14 juveniles/ m2. Some tanks also held a variety of othe r aquatic animals, including other syngnathids ( n = 10), non-syngnathid fishes ( n = 8), and invertebrates ( n = 12). Twenty-two tanks housed only Hippocampus spp. Health and reproduction records Annual m ortality ranged from 0 to 100 %, with an average of 22.7 + 4.5 %. Sixteen tanks held seahorses that mated and produced broods; of these, breeding frequency ranged from 0.1 to 6 broods per pair per year, with a mean of 1.6 + 0.3. Total power SPLs of tanks containing adult males and females that produced at least one br ood were not significantly different than total power SPLs of tanks that did not produce broods at either the middle of the water column (123.2 + 1.2 vs. 125.4 + 1.0 dB respectively, p = 0.143) or the tank bottom (130.9 + 1.3 vs. 132.8 + 2.6 dB respectively, p = 0.231), but sample sizes were small (15 and 6 tanks, respectively). Sound Analysis Mid-water level recordings At the m iddle of the water column, spectrum level SPLs ranged from 54.6 to 123.9 dB across the frequency range (Figure 2-3a, Table 24). Spectrum levels were highest from 10-30 Hz, with a mean spectrum level SPL of 109.0 dB Between 40 to 100 Hz, spectrum level SPLs averaged 93.6 dB. As frequencies increase fr om 200 to 980 Hz, spectrum level SPL gradually declined from 84.0 dB to 71.8 dB. Total power of tanks ranged from 116.3 to 142.9 dB, with a mean of 126.1 + 0.8 dB. Peak frequencies were very low, predominantly located between 7.8 and 25.5 Hz (interquartile range), with a median of 9.8 Hz. Peak amplitudes ranged from 107.4 to 140.2 dB, with a mean of 120.1 + 0.9 dB.

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38 Bottom level recordings At the bottom of the tanks, spectrum leve l SPLs ranged from 57.2 to 129.8 dB across the frequency range (Figure 2-3b, Table 2-4). Spectrum level SPLs were highest between 10 and 30 Hz with a mean SPL of 113.3 dB. Between 40 and 100 Hz, spectrum level SPLs averaged 104.4 dB. Spectrum level SPLs dropped precipitously from 102.0 dB at 100 Hz to 81.6 dB at 500 Hz, and leveled off to 77.3 dB by 980 Hz. Total power of tanks ranged from 122.3 to 146.1 dB, with a mean of 133.7 + 1.1 dB. Peak frequencies were higher th an in the middle of the water column, predominantly located between 9.8 and 56.8 Hz (I Q range), with a median of 54.8 Hz. Peak amplitudes ranged from 113.4 to 144.5 dB, with a mean of 127.8 + 1.3 dB. Wild ambient noise recordings Ten sites had a m edian Beaufort number of 2 (range 1-3) and a mean depth of 3.9 + 0.5 m (range 1.2 to 6.7 m). Spectrum level SPLs rang ed from 64.9 to 121.9 dB across the frequency range (Figure 2-3c, Table 2-4). Spectrum level SPLs were highest at 10 Hz with a mean SPL of 105.3 + 4.2 dB. Spectrum level SPLs declined most precipitously as frequencies increased to 100 Hz, at which point spectrum level SPLs averaged 86.1 dB + 3.3 dB, then further declined gradually to 76.1 + 1.5 dB by 980 Hz. Total power ranged from 103.3 to 132.6 dB, with a mean of 119.6 + 3.5 dB. Peak frequencies were very low, predominantly located between 7.1 and 13.5 Hz (IQ range), with a median of 9.3 Hz. Peak amplitudes ranged from 87.2 to 123.4 dB, with a mean of 109.0 + 4.4 dB. Ambient Noise Comparisons Total power SPLs of bottom -level recordings were significantly louder than mid-level recordings among aquaria ( p < 0.0001). Total power SPLs of ambient noise in the wild were not significantly different from total power SPLs of ambient noise in aquari a at the middle of the

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39 water column ( p = 0.095), but were significantly quieter th an total power SPLs of ambient noise in aquaria at tank bottom (p = 0.002). Effects of Design Variables on Total Power W all material, bottom habitat type, and their interaction had significant effects on total power (F6,40 = 3.69, p = 0.0052; F4,40 = 5.36, p = 0.0015; and F2,20 = 12.73, p = 0.0003, respectively; Figure 2-4). Wall material, bo ttom habitat type, and their interaction had significant effects on mid-level power (F3,21 = 3.79, p = 0.0256; F2,21 = 11.03, p = 0.0005; and F1,21 = 23.04, p < 0.0001, respectively), whereas only bottom habitat type had significant effects on bottom-level power (F2,21 = 5.43, p = 0.0126). For mid-level recordings, glass tanks were significantly louder (at a mean of 128.4 dB) than acrylic (123. 6 dB) and concrete (122.5 dB) tanks, but not fiberglass (126.4 dB) tanks. For mid-level and bottom recordings, bare-bottom tanks (at a mean of 131.0 dB at mid-level and 139.7 dB at bottom) were significantly louder than gravel-bottom (125.0 dB and 132.2 dB respect ively) and plenum (122.4 dB and 130.2 dB respectively) tanks. Among the simple effects contributing to the interaction effect of wall material*bottom habitat type at mid-level recordings, one bare bottom tank at 139.9 dB was significantly louder than gravel bottom tanks at 120.9 + 1.2 dB when tank walls were composed of acrylic (F1,21 = 47.61, p < 0.01). There was significant variation in mid-level power among wall material types of tanks with gravel bottoms (F3,21 = 8.00, p < 0.01, Figure 2-5). There was significant variation in mid-level power among wall material types of tanks with bare bottoms (F1,21 = 21.46, p < 0.01). Here, one acrylic tank at 142.9 dB was louder than one glass tank at 130.7 dB and six fiberglass tanks at 129.1 + 0.7 dB (there were no bare bottom tanks with concrete walls).

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40 Discussion These results dem onstrate a wide range of ambi ent noise to which seahorses are exposed in public aquaria. The loudest tanks demonstrate to tal power SPLs that are 4.4 and 3.9 times louder than the quietest tanks at the middle of the wa ter column and the tank bo ttom, respectively (as a 6 dB increase is a doubling of pre ssure). These results are genera lly consistent with and in the range of Bart et al.s (2001) ta nk data, despite the fact that the tanks measured in the Seahorse Sound Survey were smaller overall. Noise at the bottom of the tank was significan tly louder than in the middle of the tank. This pattern was also documented in Wysocki et al.s (2007) thorough ch aracterization of the ambient noise field in a r ectangular tank, but is in c ontrast to Bart et al.s (2001) results that showed no significant differences among tank recordi ng locations. However, Bart et al.s (2001) hydrophone never touched any surfaces, whereas in this study, bottom recordings were taken with the hydrophone touching the bo ttom habitat of the tank. Ambi ent noise was studied at both mid-level and bottom surface locations to (1) de monstrate the variation in sound pressure at different locations in the tank, and (2) to generate hypotheses about the differences in ambient noise to which fish are exposed, based on their po sitioning in the water column. Fishes that are pelagic or spend most of their time in the water column are most likely to be exposed to the lower levels of sound measured in this regi on of the tank. However, there are many bottomdwelling fishes ( e.g., catfish, eels, hawkfish, jawfish, loaches, grouper, seahorses, etc. ) that spend much of their lives in contact with the substrate. Loud ambien t noise from the tank bottom might transfer efficiently through sp ines, rays, or bones in contact with the bottom habitat to the skeleton, skull, and inner ear. Sound transfers more efficientl y between materials of similar impedance; as such, solid-to-solid transfer is mo re efficient than liquid-to-solid transfer. It

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41 would be worthwhile to study this hypothesis further and to char acterize, compare and contrast perceived sound pressure levels among wate r-column vs. bottom dwelling fishes. This distinction is especially important in light of ambient noise levels in the middle of the water column of aquaria not be ing significantly different from levels in the wild, but tank bottoms are significantly louder. Thus, benthic dwelling animals such as the seahorse are chronically exposed to louder ambient noise in a tank environment than they would normally be exposed to in the wild, but the ambient noise to which mid-water column dwelling fishes are exposed in tank environments are similar to ambient noise levels in the wild. These results suggest that some ambient noise levels can be loud enough to mask hearing in fishes. Masking noise levels discussed in Chapter 1 are within the range of ambient noise encountered in the Seahorse Sound Survey. At the middle of the water column and in the seahorses best hearing range (100-400 Hz, Chapter 3), spectr um level ambient noise ranged from 60 to 107 dB, with a mean of 85 dB. At tank bottoms, spectrum level ambient noise in the same frequency window ranged from 69 to 122 dB, with a mean of 94 dB. Mean total power SPLs were also well above 100-110 dB. Furt hermore, broadband noises cause a more pronounced masking effect than do narrowband noi ses (Hawkins and Chapman, 1975), thus the broadband nature of ambient noise is of additi onal concern. Masking, th en, is likely to be common for both hearing specialis ts and generalists in many aquarium environments. Though sound production in diverse representa tive fishes among actinopterygians is widely reported, very few publications report SPLs. Table 2-5 lists spectrum level SPLs at peak or dominant frequencies of calls from several mari ne and freshwater fishes. Calls range from 90 to 126 dB SPL re: 1 Pa. Masking studies indicat e critical ratios aver aging around 20 dB; thus, calls must be at least 20 dB loude r than spectrum level ambient noise to be detected. It is clear,

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42 then, that quieter calls (from H. erectus, P. martensii, and the drumming sounds of several otophysans) are at risk of being masked by typical ambient noise in aquaria. Other species ( e.g., M. undulates, O. tau, C. nebulosus, T. vittata, B. modesta ) may be able to overcome the critical ratio in an aquarium with mean levels of ambi ent noise. However, signals can be designed in other ways to increase the likelihood of de tection. For instance, a broadband sound ( e.g., knocking or stridulation) is more likely to be detected than a sound at the same spectrum level SPL limited to a narrow frequency band (tone Egner and Mann, 2005). Conversely, maximizing peak energy within the most sens itive hearing range of fishes is another strategy evident in seahorses (Chapter 3) and gobies (Lugli et al., 2003), for example. Sudden onset (such as the seahorse stridulation, Chapter 3), repetition (such as the pulse tr ain of gobies, Barimo and Fine, 1998; Lugli et al., 2003), and incr eased duration (such as the plai nfin midshipman boatwhistle, Bass and McKibben, 2003) also increase likeliho od of detection (Dusenbery, 1992; Bass and Clark, 2003). Direct hearing tests of masking of conspecific si gnals by aquarium ambient noise (using, for example, the auditory evoked potential method) are warranted. Prolonged exposure to loud ambient noise can lead to hearing loss that persists even after noise is removed. Temporary threshold shifts (TTS) have been documented for hearing specialists ( e.g., Popper and Clarke, 1976; Scholik a nd Yan, 2001a&b, 2002b; Amoser and Ladich, 2003; Smith et al., 2004) when exposed to noise with spectrum level SPLs as low as 134 dB re: 1 Pa that persist for up to two weeks or longer, but a hearing gene ralist did not exhibit TTS when exposed to noise with a spectrum leve l of 142 dB re: 1 Pa (Scholik and Yan, 2002a). In either case, the ambient noise levels of most aquaria are lower than those required to induce TTS even among hearing specia lists; thus TTS is not likely to be of concern for fishes in aquaria.

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43 The potential for fish ear damage from e xposure to ambient aquarium noise is also unlikely. Damage was documented after exposure to 180 dB re: 1 Pa (spectrum level and total power, respectively, per Hastings et al., 1996 and McCauley et al., 2003), but not at 140 dB (spectrum level, Hastings et al., 1996). Thes e values are far above ambient noise levels encountered in the Seahorse Sound Survey. It is clear, however, that the ambient noi se encountered in some aquaria can induce a chronic stress response in fish es. As detailed in Chapter 4, I demonstrate a chronic stress response (evidenced by behavioral differences reduced growth and body condition and altered leukocyte differential profiles) among H. erectus housed in tanks with total power SPLs of 123 dB re: 1 Pa at the middle of the water column and 137 dB at the bottom. These are approximately 3 dB below and above (respective ly) mean ambient noise levels encountered among public aquaria. Chronic stress is likely endured by seahorses in many captive aquaria at or above mean ambient noise levels. Given th e high mortality rate suffered by seahorses in public aquaria (22.7%), it is critical to minimize stimuli that induce chronic stress, in order to improve health, welfare, and immunocompetence. Questionnaire data revealed a variety of system designs. The variab ility inherent in a survey of this nature may obscure trends associat ed with any one particular variable, hence, the majority of design variables did no t reveal patterns in relation to ambient noise. It is notable, however, that wall material type and bottom ha bitat type had signifi cant effects on ambient noise, despite the variability in tank/system design among surveyed tanks. My results are generally consistent with Bart et al. (2001), who found lowest SPLs among concrete tanks and highest SPLs in fiberglass tanks (acrylic and glass tanks were not measured in their study).

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44 Reverberations (described by Yost, 1994, in Ak amatsu et al., 2002, as the persistence of sound in an enclosed space as a result of multiple reflections after sound generation has stopped) and resonance (the tendency of a system to oscillate at maximum amplitudes at certain frequencies) are acoustic phenomena that have lo ng been recognized in small tanks (Parvulescu, 1964, 1967). Together, these phenomena tend to in crease sound duration, frequency distribution, and amplitude of signals (Akamats u et al., 2002). For captive fish es in aquaria, this poses two potential problems: ambient noise may be amp lified, and conspecific signals may be severely distorted, as vividly dem onstrated by Yagers (2002) recordin gs of the click of a pipid frog in pond vs. small tank environments. In effect, signal distortion poses the same problems to social behavior in fishes as does masking in small tanks, as discussed earlier. A troublesome consequence of this phenomenon is the questionabl e results it may yield for behavioral acoustic experiments in laboratory aquaria (Akamatsu et al., 2002). Based on these results and corr oborating literature, aquarists and aquaculturists are advised to choose tanks with concrete or acrylic walls, to provide subs trate, and to choose tanks with minimum resonant frequencies above the hearing range of resident fishes. Akamatsu et al. (2002) provide equations for calculating minimu m resonant frequencies of rectangular and cylindrical tanks. Based on their equations, rectangular tanks up to 4,000 L have minimum resonant frequencies above 1,000 Hz; tanks must be a volume of approximately 38,000 L before minimum resonant frequencies fall into the ra nge of 500-600 Hz. Thus, resonance is not likely to be a problem in small tanks for hearing generalist fishes, that demonstrate lowest hearing thresholds below 500 Hz (Popper et al., 2003). In contrast, for hearing sp ecialists, some of which have sensitive hearing up to 2000 Hz and less sensitive hearing up to 4,000 Hz (Popper et al., 2003), rectangular tanks as small as 570 L may have minimum resonant frequencies that fall

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45 into their range of best hearing sensitivity ( 2,000 Hz), with very small tanks (38 to 570 L) exhibiting minimum resonant frequencies in th e range of 2,000 to 4,000 Hz. The point here is that tank size can be chosen to avoid resona nt noise in a fishs range of hearing. Davidson et al. (2007) make sensible recommendations to reduce sound impinging upon fiberglass tanks. Additionally, I su ggest the following, based on tria ls in my laboratory: Choose a smaller, less powerful, and thus quieter pump to perform filtration and circulation; place the pump on the floor or a surface that is not direct ly in connection with the tank (such as on the shelf of the tank stand); move noisemakers (such as airstones or powerheads) to the sump of the tank; install subsurface drains (thereby minimizi ng noise associated with water movement at the surface); and install soundproofing loops in fl exible plumbing lines (per A. Noxon, Acoustic Sciences Corporation, pers. comm.). S oundproofing loops decouple sound and vibration traveling through the water and pipe walls from the pump end to the tank end. Loops must float, they may be supported by a bungee cord tied to a wall, fo r example, not in connection with the tank, and the ends of the loops cannot touch. Finall y, furnish an aquarium with substrate, rockwork, and other habitat to ab sorb reverberant soundwaves in tanks. These common sense design modifications may reduce ambient noise in tank environments and ameliorate some consequences of loud noise exposure to tank inhabitants.

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46 Table 2-1. Spectra Plus processing settings. Scaling........ Sampling Rate (Hz)........ Logarithmic, peak amplitude 44100 Sampling Format.... 16-bit, Stereo Standard Frequency Wei ghting. Flat (none) Decimation Ratio... 22 Frequency Limit. 1002 Hz, Low-pass filter enabled FFT Size......................... 2048 Spectral Line Resolution.... 0.979 Hz Smoothing Window... Hann Averaging Settings. Infinite, Linear, Disable Peak Hold FFT Overlap... 0 Time Resolution. 1.022 seconds Input Signal Overload........................ Enable Overload Detection Exclude Overloaded Data from Processor Table 2-2. Public aquarium particip ants of the Seahorse Sound Survey Dallas World Aquarium. Kingdom of the Seas Aquarium. Maritime Aquarium The Aquarium at Moody Gardens. New England Aquarium. Pittsburgh Zoo and PPG Aquarium... Ripleys Aquarium of the Smokies Riverbanks Zoo.. Tennessee Aquarium.. Dallas, TX Omaha, NE Norwalk, CT Galveston, TX Boston, MA Pittsburgh, PA Gatlinburg, TN Columbia, SC Chattanooga, TN

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47 Table 2-3. Frequency distributi on of tank design specifications and animal inhabitants. n represents number of tanks. Tank Dimensions, Materials, and Habitat n Aeration and Drainage n Life Support Equipment n Other Noise Sources & Animal Collection n Tank Shape Rectangle Other polygon Bow-shaped Cylinder Bubble Crescent Hexagon Tank Wall Material Fiberglass Glass Acrylic Concrete Stand Material Plastic Wood Metal Concrete Bottom Habitat Gravel Bare Plenum 31 3 2 2 1 1 1 14 13 11 3 15 13 9 4 26 9 6 Aeration Remote Blower Air Pump Unreported # Air Endpoints One Two Four Air Endpoint Type Open End Airstones # Drains None One Two Three Drain Type Surface Skimmer Subsurface 17 3 2 13 8 1 12 10 5 29 6 1 32 4 Heater Installation Plumbed In-Line Sump Tank Chiller Installation Plumbed In-Line Sump # Pumps One Two Three 1st Pump Installation PVC Pipe Tank Submersion Sump Submersion Vinyl Tubing 2nd Pump Installation PVC Pipe Vinyl Tubing Tank Submersion 3rd Pump Installation PVC Pipe 12 11 6 2 2 18 19 2 35 2 1 1 14 6 1 2 Other Noise Sources Pumps TV/Audio Chillers Drainage Overflows Fans Air Piston Animal Collection H. erectus H. reidi H. abdominalis H. fuscus H. subelongatus H. zosterae H. kuda H. procerus Other sygnathids Non-sygnathid fishes Invertebrates 24 4 2 2 1 1 12 10 8 5 2 2 1 1 10 8 12

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48 Table 2-4. Descriptive statistics of spectrum level SPLs, peak amplitude, total power (all in dB SPL re: 1 Pa), and peak freq uency (in Hz). Mid-Level Recordings Bottom-Level Recordings Wild Recordings Hz Min Max Mean + SEMinMaxMean + SEMinMaxMean + SE 10 88.67 123.75 113.23 + 1.3192.27129.81115.22 + 1.4982.07121.91105.29 + 4.18 20 94.21 123.88 107.92 + 1.28100.80129.18111.93 + 1.2674.67118.7898.07 + 5.37 30 91.40 121.02 105.93 + 1.28101.65129.11112.66 + 1.2673.21115.8094.77 + 5.24 40 82.76 107.28 97.67 + 0.8993.83119.29106.24 + 1.1068.03110.9792.89 + 4.88 50 82.76 116.25 97.95 + 1.1595.82125.90108.00 + 1.4469.12108.3390.35 + 4.19 70 75.74 107.83 93.24 + 1.1890.61120.84105.09 + 1.4166.79103.6187.67 + 3.79 80 76.15 110.69 91.33 + 1.3489.81120.55103.05 + 1.3765.75101.4686.78 + 3.61 90 73.54 106.15 90.50 + 1.3289.27118.25101.96 + 1.3066.24100.9187.13 + 3.25 100 76.10 105.51 90.70 + 1.2585.40121.75101.94 + 1.3664.9099.0986.05 + 3.34 200 66.44 106.98 83.98 + 1.4278.62118.2794.61 + 1.3967.4296.5782.59 + 2.90 400 60.34 102.35 80.14 + 1.7269.45117.6486.76 + 1.7970.5489.7580.33 + 2.33 500 60.83 101.68 76.57 + 1.6564.07111.2981.62 + 1.8270.8588.1879.68 + 1.98 700 54.55 91.70 73.66 + 1.6057.65105.1480.27 + 1.6769.6084.0876.97 + 1.63 800 56.97 97.42 72.58 + 1.6557.66104.2278.37 + 1.8570.4683.3977.05 + 1.33 980 54.63 98.46 71.77 + 1.9457.1596.6477.30 + 1.7068.5783.6576.12 + 1.54 Peak Hz 4.89 178.14 22.96 + 5.134.89178.1445.58 + 6.494.8917.6210.18 + 1.30 Peak Amp 107.36 140.24 120.06 + 0.89113.36144.52127.82 + 1.3087.16123.39109.04 + 4.35 Tot. Power 116.34 142.86 126.09 + 0.84122.33146.05133.68 + 1.06103.34132.62119.57 + 3.45

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49 Table 2-5. Spectrum-level SPLs at peak or domin ant frequencies of representative fish sounds. Some values reported as mean + SE. Species Call Peak Hz SPL (dB re: 1 Pa) Dist (cm) Reference Marine Species Hippocampus erectus Stridulation 232 + 16 94 + 1 7.5 P. Anderson and D. Mann, forthcoming Micropogonius undulatus Pulse train 460 114 100 Barimo and Fine, 1998 Cynoscion nebulosus Pulse Train 350 120 5 Locascio and Mann, 2008 Opsanus tau Grunt 134-170 123 + 2.3 100 Barimo and Fine, 1998 Opsanus tau Boatwhistle 230-270 126 + 1.2 100 Freshwater Species Corydoras paleatus Stridulation 2000 85 5 Ladich, 1999 Padogobius martensii Pulse Train Tone 67-104 120-200 90 98 5-10 5-10 Lugli et al., 2003 Serrasalmus nattereri Drum 200 95 5 Ladich, 1999 Pimelodus pictus Stridulation Drum 1500-3000 100 102 105 5 5 Agamyxis pectinifrons Stridulation Drum 800-1500 100 105 90 5 5 Pimelodus blochii Stridulation Drum 2000 200-400 105 97 5 5 Platydoras costatus Stridulation Drum 400-800 100 110 105 5 5 Gobius nigricans Tone 73-103 113 5-10 Lugli et al., 2003 Botia modesta Knock 100-300 120 5 Ladich, 1999 Trichopsis vittata Pulse train 1500 124 3 Wysocki and Ladich, 2001

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50 Figure 2-1. The Seahorse Sound Survey kit.

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51 Figure 2-2. Acoustic sampli ng sites in Tampa Bay, FL.

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52 Figure 2-3. Power spectra from representativ e tanks at the minimum, maximum (both in gray) and median (in black) of the range. a = Recordings from the mi ddle of the water column, b = recordings from tank bottom, c = recordings from Tampa Bay. a b c

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53 105 110 115 120 125 130 135 140 145Concrete A cryl i c F i berg l ass G lass Wo od C o n cr e t e Metal P last i c P l e nu m G rav el B a r eWall Material Stand Material Bottom HabitatSPL (dB re: 1 uPa)A A A,B B CC D E E F Figure 2-4. Total power vs. sele cted design specifications. Mid-level SPLs in white, bottomlevel SPLs in gray. Error bars are + SE. Letters denote si gnificant differences among treatments within a subgroup (Tukeys test, p < 0.05). 115 120 125 130 GlassFiberglassConcreteAcrylic Wall Material TypeSPL (dB re: 1 uPa) A B,C A,B C Figure 2-5. Mid-level total power vs. wall material type among tanks with gravel bottoms. Error bars are + SE. Letters denote significant di fferences among wall material types (Tukeys test, p < 0.05).

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54 CHAPTER 3 AUDITORY EVOKED POTENTIAL OF T HE LINED SEAHORSE, HIPPOCAMPUS ERECTUS, AND ITS RELATIONSHIP TO CON SPECIFIC SOUND PRODUCTION Introduction The auditory evoked potential (AEP) technique to m easure hearing ability is widely practiced among human clinicians ( e.g., Davis, 1976; Picton et al., 1981; Schroeder and Kramer, 1989) and has been expanded to test hearing ability of representati ves from many vertebrate taxa (Corwin, 1982), including fish (Kenyon et al., 1998). Among fishes, this method has advantages over behavioral methods such as cardiac suppression ( e.g., Chapman and Sand, 1974), ventilatory suppression ( e.g., Fay, 1995), stereotyped defense responses ( e.g., Kenyon, 1996), classical conditioning (Fay, 1988a), instrumental avoidance conditioning (e.g., Tavolga and Wodinsky, 1963), and operant conditioning (e.g., Yan and Popper, 1993). Behavioral methods pose various drawbacks, includi ng inconsistency of response, ex cessively long training periods, and stressful stimuli (Kenyon et al ., 1998). In contrast, AEP is a non-invasive far-field recording of synchronous neural activity in the eighth nerve and brains tem auditory nuclei elicited by acoustic stimuli (Jacobson, 1985). The objectives in this study are two-fold. First, I aim to characterize the hearing ability of the lined seahorse (Hippocampus erectus) in a comprehensive manner by measuring both the pressure and particle acceleration of the acoustic stimuli in hearing tests. These components of sound contribute in different ways in the nea r-field and far-field of sound sources. A vibrating sound source produ ces two physical changes in the surrounding environment: particle motion and pressure wave propagation of the surrounding medium (Dusenberry, 1992). The near-field of a sound source is dominated by local hydrodynamic fl ow, established by the displacement of water molecules (particle moti on) adjacent to the sound source (Bass and Clark,

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55 2003). In the far-field, propagating waves begin and can generate a pressure wave in addition to particle motion. The inner ears of fishes include three otolithic end organs (the saccule, utricle, and lagena) containing a calcium-carbonate otol ith, encased within a sac lined with a sensory epithelium. The sensory unit of the epithelium is the neuromast, or hair cell. It is a mechanosensor that detects particle motion. When the particle motion component of a propagating sound wave approaches a fish, the same degree of particle mo tion is generated in the watery tissues of the fish as in the surrounding medium. The otolo lith, however, moves at a different amplitude and phase due to its greater density. This sets up a relative displacement between the otolith and the neuromasts that is proportional to acoustic particle motion (Popper and Fay, 1999). The gas bladder, thought to have originally evolved as a mechanism for buoyancy control, incidentally affects the hearing sensitivity of fishes. When a volume of gas is exposed to oscillating pressure changes, it will display la rger volume pulsations than a comparable volume of water. So, it acts as an am plifier, converting pressure fluctu ations to motions detectable by the ears. This enables so me fishes to detect the pressure co mponent of sound as well, especially hearing specialist fishes, or fishes which have evolved a bony or gaseous vesicular connection between the gas bladder or inne r ear (Popper et al., 2003). It is generally well-accepted that hearing specialist fishes respond to both particle acceleration and pressure, but are mo re sensitive to pressure partic ularly in the far-field and at frequencies above 70 Hz (Fay et al., 1982). Hearing generalist fi shes, that have no specialized connections between the swimbladder and inner ea r, have yielded equivoc al data concerning the relative importance of pressure sensitivity to sound detection and processing (Cahn et al., 1968; Sand and Enger, 1973; Chapman and Johnstone, 1974; Fay and Popper, 1975; Jerk et al., 1989;

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56 Lovell et al., 2005). The consensus that might be drawn from this literature is that both acoustic modalities may be detected and processed by hearing generalist fishes, though the relative contributions of each may vary with respect to distance, frequency, and sound pressure level; both modalities are thus reported here. I also aim to characterize conspecific s ound production and to draw inference about audition of these sounds. Seahorses produce clicks that were first desc ribed by Fish (1953) and later expanded upon by Colson et al. (1998). Clicks are short duration, broadband sounds (Zelick et al., 1999) that, am ong seahorses, are characterized by a peak frequency that is inversely related to body size (Colson et al., 199 8). The seahorse click is a stridulatory sound produced from the bony articulation between the supraoccipital and the coronet (Fish, 1953; Colson et al., 1998) and is demonstrated in ma ny behavioral contexts, including feeding (Colson et al., 1998), aggression and competition for mates (Vincent, 1994b), distress (Fish, 1953), and stress (Chapter 4). Audition of conspecific clicks must be demons trated as a precursor to testing hypotheses of conspecific acoustic communication. Materials and Methods Animal Accession, Holding, and Husbandry Procedures Lined seaho rses (H. erectus) were collected as bycatch from shrimp trawl nets and donated by local fishermen. Upon accession, animals were quarantined for one month prior to transfer to a sound-dampened holding system. Clear roun d acetate tags (approx. 1 cm diameter) were marked with alphanumeric codes, hung on monof ilament line collars, and tied around the necks of seahorses (tagging methods modified from Vincent and Sadler, 1995) Animals were fed frozen mysids (Piscine Energetics) in the mornings and live Artemia sp. enriched with Roti-Rich in the afternoons. Tanks were siphoned clean of debris twice daily and sy stem water changes of 10% were performed weekly. Water quality para meters remained within the following ranges

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57 during holding: Temperature, 25-27C; sa linity, 28.5 to 31.5 ppt; ammonia-nitrogen, 0 ppm; nitrite-nitrogen, 0 ppm; nitratenitrogen, 2.8-22.7 ppm. Eleven animals were transferred to a sound-proof ed tank with an estab lished biofilter 2 to 11 days prior to testing. Soundproofing was accomp lished by resting the frame of the tank on a sturdy lab bench with sections of bearing felt, in stalling a subsurface drain that transferred water to a sump resting on the floor where filtration occurred, and using a quiet 15W water pump with a flexible return pipe that re turned water to the tank below the water surface. A loop was suspended in the flexible return line; this a ttenuated vibration and sound traveling through the return water and pipe walls (A. Noxon, Acoustic Sciences Corp., personal communication, Figure 3-1). The ambient noise profiles of both the holding sy stem and the sound-proofed tank were measured with an HTI-96-min hydrophone (Hi gh Tech Instruments, Inc., sensitivity = 164.1 dB re: 1V/Pa, bandwidth = 2-30,000 Hz), for sound pressure level (SPL) measurements. The ambient noise profile of the sound-proofed tank was also measured with an Acoustech geophone probe (Acoustech Corporation, sensitiv ity = 212 V/m/s, bandwidth = 100-1000 Hz) for measurements of particle motion. Both instruments, when in use, were connected to the line-in port of a laptop computer running CoolEdit (Synt rillium Software). Hydrophone recordings were collected from the middle and bottom of ta nks. Geophone recordings were collected from the bottom of tanks in the center, along three or thogonal axes, because particle motion is a vector quantity (as opposed to pressure, which is a scalar quantity). Resulting sound files were calibrated according to manufactur er instructions and post-proce ssed with SpectraPlus (Pioneer Hill software). Analysis settings used in SpectraPlus are summarized in Table 3-1. The Acoustech geophone probe measures particle velocity. To convert to acceleration, Fast-Fourier

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58 Transforms (FFTs) processed by SpectraPlus were exported in to a spreadsheet program and particle velocity values c onverted to particle accelerati on using the following formula: Acceleration (m/s2) = v2 f (3-1) where v = velocity (m/s), = pi (~3.14), and f = frequency (Hz) (Casper and Mann, 2006). The magnitude of particle accelerat ion was calculated by vector aver aging according to the following equation: 222zyxon Accelerati (3-2) where x, y, and z refer to acceleration (m/s2) in each of three orthogonal axes. AEP Experimental setup The testing cham ber consisted of a stee l tube (1.22 m high, 20.32 cm diameter, 0.9525 cm thickness), closed at the bottom with a square steel plate (60.96 X 60.96 cm), and oriented vertically. Four anti-vibra tion floor mounts (Tech Products Corp., 51700 Series) were placed under each corner of the base of the tank. The tu be was filled with saltwater of approximately 26C up to a height of 1.12 m. A laboratory stand was supported on an adjacent vibrationisolated table and scaffolding descended into the tube for animal suspension. A University Sound UW30 speaker was placed at the bottom of the tube in the center. This setup was enclosed inside an audiology booth. For testing, individual fish were secured in a harness constructed from Nitex mesh, fastened with clamps to scaffolding 2.5 cm be low the water surface. The harness restricted movement while allowing normal respiration. Subdermal stainless steel needle electrodes (Rochester Electro-Medical) were used to record the AEP signal. An electrode was inserted about 1 mm into the head, over the medulla region. Reference and ground electrodes were

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59 placed directly in the water in close proximity to the fish. Evoked potentials recorded by the electrode were fed through a Tucker Davis Tech nologies (TDT) HS4 fiber obtic headstage and into a TDT RP 2.1 processor, routed into the co mputer and averaged by TDT BioSig software. All eleven seahorses were tested for AEPs to tone stimuli (described below); also, a dead goldfish (Carassius auratus) was run to generate control AEP signals. Sound stimuli and AEP waveform recordings were produced with a TDT AEP workstation running SigGen and BioSig software. Sounds were generated by an RP 2.1 Enhanced RealTime processor, fed through a PA5 programmable attenuator to cont rol sound level, and amplified by a Hafler Trans.Ana P1000 110 W profe ssional power amplifier before being sent to the UW30 speaker, where sound was emitted (Figure 3-2). Sound generation, calibration, and AEP acquisition Calibration Acoustic stimuli were calibrate d with the HTI-96-min hydrophone for pressure measurements of tones and the Ac oustech geophone in three orthogonal axes for particle motion, all connected to the RP 2.1 processor. Duri ng calibration, the hydrophone or geophone was positioned in the experimental set up in place of the fish at the level of the animals head, and the stimulus presentation prot ocol as described for AEP acquisition (below) was executed, except without phase alternation. Signals were captu red and averaged by BioSig. Resulting time domain averaged signals were ex ported as ASCII formatted files, imported in SpectraPlus, and 4096 point FFTs run (SpectraPlus settings in Table 3-1) to generate power spectra, from which peak amplitude measuremen ts were taken. Particle acceleration was calculated per Equations 3-1 and 3-2. Calibration runs were conducted daily. AEP acquisition. Stimuli consisted of 60 ms pulse d tones gated with a Hann window. The phase of the tone was alternated between pr esentations to minimize electrical artifacts from the recordings. During each trial, 9 differe nt frequencies were presented: 100, 200, 300, 400,

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60 600, 800, 1000, 1500, and 2000 Hz. Amplitudes at each frequency were presented within a range of approximately 74 to 148 dB re: 1 Pa and 8.60 X 10-6 to 0.67 m/s2, beginning at amplitudes below threshold and increasing in 6 dB steps until a threshold was visually detected in the digital signal output (see Data Analysis). Post-hoc trials were run at amplitudes that were 3 dB below visual threshold to increase the accuracy of threshold determination. Up to 2,000 signal presentations (or until detection was visu ally confirmed) were averaged to measure the evoked response at each level of each frequency. Data analysis Evoked potential traces were tr ansform ed with the Hann window function and converted to power spectra with a 2048-point FFT in BioSig. Evoked potentials are visualized as peaks that occur at twice the frequency of the presented stimu lus (Figure 3-3). This is a well-established phenomenon in evoked potentials of fish to pur e tones in the freque ncy domain (Egner and Mann, 2005). Visualized peaks were considered true evoked potentials if they were at least 3 dB above the average of all peaks o ccurring within a window of 50 Hz above the presented stimulus frequency. AEP thresholds were defined as the lowest amplit ude at which a true evoked potential, according to these crite ria, was visualized. AEP wave forms of live seahorses were checked against AEP waveforms of dead goldfish to ensure that the identified peak was not a stimulus or electrical artifact. Click Recordings Experimental setup A rectangular polystyrene fish shipm ent box with inner dimensions of 37.7 cm L X 37.7 cm W X 19 cm H and wall thickness of 2.3 cm was marked with a 5 cm X 5 cm square grid on the floor of the box, placed on the floor, and filled to 17 cm high with saltwater. The Acoustech probe was placed against one of the walls of the box at its center, with the motion sensitive axis

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61 oriented perpendicular to the wall (facing toward the center of the box, Figure 3-4). I utilized the Acoustech probes incorporated hydrophone (sens itivity = -176 dB re: 1 V/Pa, bandwidth = 10 1,000 Hz) for pressure sensitive measurements and the geophone for particle motion sensitive measurements. The hydrophone was fed to the le ft-channel and the ge ophone to the rightchannel of the line-in port of a laptop com puter running CoolEdit (Syntrillium Software) for recording at a sampling rate of 44.1 kbps. The recording system of the laptop was previously calibrated with a 1.0 Vpeak sine wave. One to three seahorses were placed inside th e box at any one time. An aliquot of live Artemia sp. was added to the box to stimulate a feeding response. Recording began when the first seahorse made its first feeding strike. Du ring recording, as each animal struck, the animal ID, distance to the Acoustech geophone, and th e timestamp of the strike was recorded. Recording ceased after 30 min or earlier if animals stopped feed ing for an excessive period of time. Data analysis Signal processing and analysis. Using CoolEdit, documented clicks from resulting .wav files were isolated within the center of 5 s windows, copied a nd pasted into new, individual sound files for each click. The first two seconds of each click file were used to obtain a noise profile using a 4096 pt FFT. This noise profile was used to reduce noise in the entire click file by 40 dB. Noise-reduced files were then calibrated for voltage in SpectraPlus according to manufacturer instructions and post-processed. C lick analysis settings us ed in SpectraPlus are summarized in Table 3-1. For both the pressure-sensitive waveform (left-channel) and the particle-motion sensitive waveform (right-channel), I took the following cursor measurements in the time domain:

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62 Duration: Duration of entire click waveform (in milliseconds) Rise: Time from onset of waveform to largest non-resonant peak (in milliseconds) Fall: Time from largest non-resonant peak to end of waveform (in milliseconds) Peak Amplitude: Amplitude at largest non-re sonant peak (in dB SPL (re: 1 Pa) or m/s)2 Peak-to-Peak Amplitude: Amplitude differen tial from largest non-re sonant positive peak to largest non-resonant ne gative peak (in dB SPL (re: 1 Pa) or m/s, Figure 3-5)2 For both waveforms, I took the following curs or measurements in the frequency domain, examining a window from 2-998 Hz: Frequency at 25% Peak Amplitude: On both sides of the broadband signal (in Hz) Peak Frequency: Frequency of 1st, 2nd, and 3rd highest amplitude peaks (in Hz) Peak Amplitude: Amplitude of 1st, 2nd, and 3rd highest amplitude peaks (in dB SPL (re: 1 Pa) or m/s2)2 Total RMS Power: From 2 to 998 Hz (in dB SPL (re: 1 Pa) or m/s2, Figure 3-6)2 Statistical analysis. Clicks were categorized from visual inspection as either resonant or non-resonant, characterized by the presence or absence (respectively) of a series of high amplitude, high frequency waveforms occurring duri ng or immediately after the rise of the click in the time domain. For each measure, descriptiv e statistics were computed for total, resonant, and non-resonant clicks from each animal a nd for summed clicks, for both pressure and acceleration. F-tests were run between non-resona nt and resonant clicks for all measures to examine heterogeneity of variance. t-tests (assuming equal variances only where F-tests were non-significant) were run between n on-resonant and resonant clicks for all measures to examine differences in click characteristics in the time and frequency domains. Paired t-tests were run between pressure-sensitive waveforms and par ticle-motion sensitive waveforms to examine 2 The Acoustech geophone probe measures particle velocity In the time domain, particle motion measurements are represented in terms of particle velocity. In the frequenc y domain, particle motion measurements are represented in terms of particle acceleration, and calculated using Equation 3-1.

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63 differences in the time domain and in peak frequencies. Finally, a repeated measures ANOVA model was employed using SAS (The SAS Institute) to examine possible differences in peak frequencies, peak-to-peak amplitudes and cl ick durations, between the sexes and among individuals, for both pressure-sensitive wavefo rms and particle-motion sensitive waveforms, according to the following model: y = sex + animal(sex) + (3-3) where sex is a fixed factor--male or female, an imal(sex) is a random factor referring to the individual animal (nested within sex), and is the error term. Data were not balanced, so Type III Sums of Squares were used. Results Ambient Noise The long-term holding tanks in which anim als were housed prior to transfer to the AEP laboratory demonstrated an average total RMS power (within the 2 to 998 Hz frequency range) of 117.4 + 0.9 dB SPL (re: 1 Pa) at the middle of the water column and 128.8 + 1.4 dB at the bottom (Figure 3-7a). The soundproofed AEP labor atory tank demonstrated an average total RMS power of 115.8 + 0.5 dB at the middle of the water column and 120.5 + 0.2 dB at the bottom (Figure 3-7b), and a vector-a veraged total RMS power of 4.58 X 10-3 m/s2 (Figure 3-8). AEP The AEP audiogram s averaged from 11 H. erectus are plotted in Figure 3-9. For sound pressure, this species most sensitive hearing range is below 400 Hz, with a minimum threshold of 105.0 + 1.5 dB SPL re: 1 Pa at 200 Hz (mean + SE). After 600 Hz, hearing thresholds increase to levels above most environmentally relevant noise (Uric k, 1975). For particle acceleration, this species most sensitive he aring range is below 300 Hz, with a minimum threshold of 3.46 X 10-3 + 7.64 X 10-4 m/s2 at 200 Hz.

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64 Click Recordings I characterized up to ten clicks from ten seahorses, for a total of 85 clicks. Pressure Characterization. Seahorses produced clicks at an average distance of 7.5 + 0.4 cm from the center of the Acoustech geophone. C licks had an average duration of 109.8 + 7.6 ms, with a rise time of 48.5 + 5.2 ms and a fall time of 59.9 + 3.7 ms. 71.7% of clicks presented peaks in the positive phase, and 28.2% in the negative phase Clicks averaged peak amplitudes of 136.8 + 1.0 dB SPL (re: 1 Pa) and peak-to-peak amplitudes of 141.6 + 1.0 dB in the time domain. In the frequency domain, the power distribution was broad, with amplitudes at 25% of peak beginning at 89 + 5 Hz and ending at 741 + 21 Hz. Peak frequency averaged 232 + 16 Hz, with an average peak amplitude of 94.3 + 0.9 dB SPL (re: 1 Pa). The most prominent peaks (I selected three peaks with th e highest amplitudes from each click) were found within an interquartile range of 166 to 343 Hz, with a mean of 271 + 10 Hz. Amplitudes of these peaks were within an interquart ile range of 86.2 to 97.3 dB dB SPL (re: 1 Pa), with a mean amplitude of 92.5 + 0.5 dB. Resonant vs. non-resonant clicks. Pressure waveforms were particularly susceptible to resonance artifacts, a comm on phenomenon in small tanks (Parvulescu, 1964, 1967; Yager, 1992; Akamatsu et al., 2002). Clicks were judged as resonant if a series of high amplitude, high frequency waveforms occurred during or immediat ely after the rise of the click in the time domain (Figure 3-10). Clicks whose resonanc e masked the peak (and the delineation between rise and fall time) were not included in analysis of measures in the time domain, but in most cases, resonance occurred after the peak and duri ng the fall time of the click. There were no significant differences in the duration, rise time, or fall time between resonant and non-resonant peaks. However, resonant peaks dem onstrated louder peak amplitudes (140.1 + 1.6 dB vs. 134.7

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65 + 1.2 dB, p = 0.009) and peak-to-peak amplitudes (144.8 + 1.4 dB vs. 139.6 + 1.2 dB, p = 0.008) in the time domain waveform. This trend is corroborated in the fre quency domain; resonant clicks had higher peak amplitudes (96.7 + 1.3 dB) than non-resonant clicks (92.4 + 1.2 dB, p = 0.015). Particle acceleration Clicks m easured from the particle accelerat ion recordings had an average duration of 154.3 + 10.7 ms, with a rise time of 55.2 + 5.4 ms and a fall time of 100.8 + 6.9 ms. 58.8% of clicks presented peaks in the positive phase, and 41.2% in the negative phase. Clicks averaged peak velocities of 1.06 X 10-4 + 1.65 X 10-5 m/s and peak-to-peak velocities of 1.91 X 10-4 + 2.96 X 10-5 m/s in the time domain. In the frequency domain, the power distri bution was also broad, with amplitudes at 25% of peak beginning at 81 + 3 Hz and ending at 681 + 17 Hz. Peak frequency averaged 265 + 22 Hz, with a peak acceleration of 1.52 X 10-3 + 1.87 X 10-4 m/s2. The most prominent peaks (I selected three peaks with the highest amplitude from each click) were found within an interquartile range of 148 to 372 Hz, with a mean of 290 + 13 Hz. Amplitudes of these peaks were within an interquartile range of 4.90 X 10-4 to 1.33 X 10-3 m/s 2, with a mean acceleration of 1.20 X 10-3 + 8.99 X 10-5 m/s 2. Comparisons between pressure waveforms and particle acceleration waveforms In the time domain, particle motion wave forms exhibited longer overall duration (p = 1.65 X 10-8), as well as longer rise (p = 2.50 X 10-4) and fall (p = 2.37 X 10-8) times. In the frequency domain, pressure waveforms tended to have mo re energy distributed at higher frequencies; frequencies at 25% of peak amplitude to the righ t of the largest peak were higher in pressure motion waveforms (p = 1.98 X 10-4). However, there were no significant differences in the frequencies of either the highest amplitude peak (p = 0.180) or the frequencies of the three highest amplitude peaks combined (p = 0.208) between the two waveform types.

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66 Comparisons among individuals and between sexes The sex of the animal had no effect on click duration (pressure, F1,8.18 = 0.39, p = 0.547; particle motion, F1,8.30 = 1.95, p = 0.199), peak-to-peak amplitude (pressure, F1, 8.97 = 1.35, p = 0.276; particle motion, F1, 75 = 0.11, p = 0.745), or peak frequency (pressure, F1, 10.35 = 0.17, p = 0.686; particle motion, F1, 10.344 = 3.70, p = 0.082) of waveforms, although sample sizes were small (n = 7, n = 3). Clicks differed significantly am ong individuals in duration (pressure, F8,75 = 15.76, p < 0.0001; particle motion, F8,75 = 9.52, p < 0.0001) and peak-to-peak amplitude of both waveforms (pressure, F8,69 = 6.36, p < 0.0001; particle motion, F8,75 = 3.58, p = 0.0015), but not in peak frequency (pressure, F8,75 = 1.29, p = 0.261; particle motion, F8,75 = 1.29, p = 0.260). Discussion My results d emonstrate that H. erectus is a hearing generalist. It s hearing sensitivity falls within the range of sensitivities documen ted for other hearing generalist fishes (e.g., Kenyon et al., 1998; Yan, 2001; Scholik and Yan, 2002a; Lugli et al., 2003; Egner and Mann, 2005; Lovell et al., 2005; Casper and Mann, 2006, Figure 3-11). While I had the ability to test this animals hearing sensitivity at up to 2,000 Hz (for sound pr essure), thresholds at frequencies above 600 Hz begin to rise into a range of high SPLs that animals are not lik ely to encounter in the natural environment (Urick, 1975). My conclusion that this seahorse is a hearing generalist is corroborated by the lack of specialized connections between the gas bladder and the inner ear (P. Anderson, pers. obs.), that is generally a requirement for h earing specialist fish es (Popper et al., 2003). There is remarkable similarity between the shapes of the audiograms for sound pressure and particle acceleration. Despite the fundamental differences betw een the pressure and particle motion component of sound, and the fundamental differences in the way each modality is

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67 processed by fishes, both hearing specialists (e.g., Ictalurus punctatus, Fay and Popper, 1975) and generalists (e.g., Ginglystoma cirratum, Casper and Mann, 2006) demonstrate similarities between the shape of audiograms for each acoustic modality. Some tests have shown dissimilarities (e.g., Hawkins and Johnstone, 1978; Kelly and Nelson, 1975) that may be due to artifactual acoustic discontinuitie s between sound pressure and particle motion in constrained testing environments. My results of seahorse click characteristics differ considerably from previous characterizations, though sample sizes in previous studies are small. Fish (1953) documented maximum energy of clicks from one female H. erectus to occur in 300-600 Hz and 400-800 Hz frequency bands. Colson et al (1998) documented peak freque ncies ranging from 1.96 to 2.37 kHz in four clicks produced by one indivi dual. The response bandwidth of Colson et als hydrophone was not stated; whether the frequenc y range of 0-1000 Hz was investigated is unknown. Even if peak frequencies above 1,000 Hz are real, they are unlikely to be of intraspecific communicative value, given the audi ogram presented here. The clicks I recorded may have contained energy above 1,000 Hz, particul arly among resonant clicks (that, in this case, may be artifactual and not a true component of the click), but given the sensitivity of H. erectus and other hearing gene ralist fishes, I considered the ba ndwidth of 0-1,000 Hz to be most worthy of investigation. The resonance artifacts I documented in sound pressure among clicks are typical of problems of sound distortion in small tanks, that have been known for some time (Parvulescu, 1964, 1967). Yager (1992) also documented resonan ce artifacts when recording clicks from the African Pipid frog (Xenopus borealis) in small tanks; showing a br oad band of high-amplitude energy between 5-40 kHz that is not present when clicks are recorded in a pond. However, my

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68 tests showed no significant differences among the frequency components of the clicks in the 01000 Hz frequency band, that is w ithin the range of hearing of H. erectus and other hearing generalists. It is likely that resonance artifacts seen in the time domain would have appeared as peaks at higher frequencies that I did not consider, as predicted by Akamatsu et al.s (2002) equations for calculating minimum resonant freque ncies, but because these peaks are outside of the hearing range of most fishes, these distor tions are not likely to be audible to them. Resonance did not alter temporal components of clicks, but resonant clicks were louder than non-resonant clicks. I hypothesize that resonance did not result in louder c licks, but rather that louder clicks were more likely to propagate standing waves in the recording chamber and generate resonance. Hearing generalist fishes may thus still be able to detect and correctly process conspecific sounds in small tanks, because resonance components that distort the original signal occur outside of the hearing range of these fishes. However, acoustic communication modalities may be hampered among hearing specialists in small tanks, where resonance components are likely to occur within their range of hearing. The seahorse click is an example of a broadband sound that increases likelihood of reception by listeners. Broadband signals are detected at lower amplitudes than tonal signals (Yost, 2000). In an environment such as a marine coastal zone where substrate, habitat, surface waves, and ambient noise serve as obstacles to sound propagation (via reflection, refraction, attenuation, masking, absorption, scattering, etc.), broadband signals promote the reliability of sound propagation where the spectral content of the signal is often di storted (Gerald, 1971). Sudden onset of the click, a temporal characteristic, also boosts likeli hood of detection (Hall, 1992).

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69 A visually striking argument for the evol ution of sound production for the purpose of conspecific communication in H. erectus is made when comparing peak frequencies of the sound with the audiogram. Because the audiogram meas ures hearing at one frequency while clicks are broadband, I adjusted the audiogram thresholds to estimate the animals ability to detect broadband sounds using a calculation proposed by Yost (2000) and employed by Egner and Mann (2005) for hearing in damselfishes. Speci fically, the audiogram was adjusted by an estimated critical bandwidth that is assumed to be 10% of the center frequency. Because sound pressure is expressed on a logarithmi c scale (dB), this adjustment is: Frequency Threshold %10log10 (3-4) Because acceleration is a linear quantity, this adjustment is: Frequency Threshold %10 (3-5) Additionally, because clicks were only charac terized for particle acceleration with the geophone in one axis, I employed audiogram thres hold measurements from only one axis with the greatest acceleration values. These comparis ons are represented in Figures 3-12 and 3-13. For both pressure and particle a cceleration, peak frequencies clus ter around the lowest threshold at 200 Hz. While only 34% of the peaks are ap parently audible in sound pressure, 93% are audible in particle acceleration. Furthermore, AEP methodology underestimates hearing thresholds in comparison to behavioral methods by up to 19 dB (Hill, 2005), so it is likely that most clicks are audible. These observa tions, coupled with the conclusion that H. erectus is a hearing generalist, lead to the conclusion that H. erectus may hear and process conspecific clicks. Correlation between auditory sensitivit y and vocalization has been documented for other sound-producing fishes as well ( e.g., Stabentheiner, 1988; Ladich and Yan, 1998; Ladich, 1999; Wysocki and Ladich, 2001; Lugli et al., 2003).

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70 Vocalization in fishes may communicate a wea lth of information, including identity (Myrberg and Riggio, 1985), sex (L adich, 1997), size (Myrberg et al., 1993), species (Spanier, 1979), location (Ladich, 1997), and behavioral state (Crawford, 1997). Among many of the generalists that create broadband sounds, modulation of the temporal component of sound (as opposed to the frequency component) is consider ed to be the most important communicative feature (Popper et al., 2003) but this is generally in reference to sounds that can vary in duration, interpulse interval of pulse trai ns, or number of vocalizations ( e.g., Spanier, 1979; McKibben and Bass, 1998). My results show no detectable di fference in the time or frequency domains between males and females, though sample sizes were small. Individual differences were detected in click duration and amplitude, that provides for the opportunity to discriminate among individuals in these measures. A seahorse may evaluate clicks produced by nearby seahorses to differentiate between mates and non-mates in populations where extrapair encounters occur (Vincent and Sadler, 1995). Likewise, an animal may click to advertise its presence or location to its mate. Clicking may also assist unpaired animals in the advertisement and location of potential mates in a sparsely di stributed population. The click is also associated with feeding strikes (Colson et al., 1998); a dvertising the presence of a f ood source to a monogamous mate may be a strategy for increasing a mates repr oductive fitness. La boratory studies have demonstrated clicking as part of an aggressive interaction between males competing for a mate (Vincent, 1994b). Ladich (1997) suggests, in Trichopsis vittata, that fish emitting louder agonistic signals may win competitive encounters; this scenario may also be plausible in H. erectus. Though correlations of signal parameters with body size were not examined in this study, Colson et al. (1998) documented a negati ve correlation between animal size and peak frequency. As seahorses exhi bit size-assortative ma ting (Foster and Vincent, 2004), the click

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71 may also assist potential mates in the selecti on of a mate of suitable size. While my study suggests that the seahorse click may have evolved to signal to c onspecifics, further studies on the function of clicking in these various contexts are warranted to test the hypotheses proposed.

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72 Table 3-1. Spectra Pl us recording settings. Ambient Noise Analysis AEP Stimulus Analysis Click Analysis Sampling Rate (Hz) 44100 48828 44100 Sampling Format 16-bit, Stereo 16-bit, Stereo 16-bit, Stereo Standard Hz Weighting Flat (non e) Flat (none) Flat (none) Decimation Ratio 11 12 11 Frequency Limit 2004.545 Hz, Low-pass filter enabled 2034.500 Hz, Low-pass filter enabled 2004.545 Hz, Low-pass filter enabled FFT Size 4096 4096 4096 Spectral Line Resolution 0.979 Hz 0.993 Hz 0.979 Hz Smoothing Window Hann Hann Hann Averaging Settings Infinite, Linear, Disable Peak Hold 1, Linear, Enable Peak Hold 1, Linear, Enable Peak Hold FFT Overlap 0% 99% 99% Time Resolution 1021.68 ms 10.07 ms 10.22 ms Input Signal Overload Enable Overload Detection Exclude Overloaded Data from Processor Enable Overload Detection Exclude Overloaded Data from Processor Enable Overload Detection Exclude Overloaded Data from Processor

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73 Figure 3-1. Soundproofed laboratory tank.

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74 Figure 3-2. AEP experiment al setup (modified from Egner and Mann, 2005). P1000 = amplifier, PA5 = attenuator, RP2.1 = processor, RFE = reference electrode, RE = recording electrode, GE = ground el ectrode, HS4 = headstage.

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75 Figure 3-3. Auditory evoked potentials (AEPs) to a 400 Hz tone pip depicted in the tim e domain (left) and in the frequency dom ain (right). a = Control AEP waveform (dead goldfish). b,c,d = AEP waveforms of H. erectus at progressively lower amplitudes. d represents amplitude at threshold. Asterisks denote AEPs that occur at twice the frequency of the presented stimulus (in this case, 800 Hz).

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76 Figure 3-4. Click recording chamber (top vi ew). The Acoustech geophone probe was placed against the wall of the chamber, with the recording axis facing toward the center. Figure 3-5. Click characteristics measured in the time domain waveform. Horizontal arrows depict time measurements, vertical arrows depict amplitude measurements. P-P = Peak to peak.

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77 Figure 3-6. Click characteristics measur ed in the frequency domain waveform. 1,2, and 3 refer to the 1st, 2nd, and 3rd highest amplitude peaks.

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78 Figure 3-7. Representative power spectra of sound pressure of (a) long-term holding tanks and (b) the sound-proofed laboratory tank. Gray = Ambient noise recorded from the middle of the wate r column. Black = Ambient noise recorded from the tank bottom. b a

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79 0.00001 0.00010 0.00100 2100198296393491589687785883981 Frequency (Hz)Acceleration (m/s^2) Figure 3-8. Power spectrum of particle accel eration of the sound-proofed laboratory tank. 100 105 110 115 120 125 130 135 140 100200300400600800100015002000 Frequency (Hz)SPL (dB re: 1 uPa)0.001 0.01 0.1 1Acceleration (m/s^2) Figure 3-9. Audiograms of the lined seahorse, H. erectus, for sound pressure and particle acceleration. Black trace = sound pressure Gray trace = particle acceleration.

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80 Figure 3-10. A resonant click, depicted in the time domain. Compare with Figure 3-5. 60 70 80 90 100 110 120 130 140 150 1002003004005006007008009001000 Frequency (Hz)Sound Pressure Level (dB re: 1 uPa) Hippocampus erectus (this study) Lepomis macrochirus (Scholik and Yan, 2002a) Opsanus tau (Yan, 2001) Padogobius martensii (Lugli et al., 2003) Astronotus ocellatus (Kenyon et al., 1998) Carassius auratus (Kenyon et al., 1998) Figure 3-11. Sound pressure audiograms of repr esentative hearing genera list fishes, measured by the AEP technique. The audiogram of a representative hearing specialist, C. auratus, is also shown for comparison.

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81 70 75 80 85 90 95 100 105 110 115 120 05001000150020002500 Frequency (Hz)SPL (dB re: 1 uPa) Figure 3-12. Comparison of the broadband sound pr essure audiogram agains t peaks of recorded clicks, represented as gray points. 0.0001 0.001 020040060080010001200 Frequency (Hz)Acceleration (m/s^2) Figure 3-13. Comparison of the broadband partic le acceleration audiogram against peaks of recorded clicks, repres ented as gray points.

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82 CHAPTER 4 SOUND, STRESS, AND SEAHORSES: THE CONSEQUENCES OF A NOISY ENVIRONMENT TO ANIMAL HEALTH Introduction Here, I examine the hypothesis that chronic ex posure to loud ambient noise acts as a chronic stressor to aquarium fish, resulting in responses among primary ( e.g., plasma cortisol), secondary ( e.g., blood glucose, hematological measures), and/or tertiary ( e.g., growth, behavior, mortality) stress indices. Stress is an important consideration in the successful husbandry of fish. Severe stress results in mortality, but even sublethal stress compromises various physiological and behavioral functions, leading to suppressed immune functio n and disease resistan ce, growth rate, and reproduction, all contributi ng to suboptimal producti on (Iwama et al., 1997). Anthropogenic noise to which wild and captive fishes are exposed is variable, but can have negative impacts at all levels of biological orga nization. Santulli et al. (1999) examined the effects of intense acoustic stimulation em itted by air guns used in seismic surveys on biochemical and physiological responses in Europ ean sea bass. Their post-shock serum analyses indicated increases in cortisol, variations in gl ucose and lactate, decreased skeletal muscle and liver ATP concentrations, and increased ADP con centrations. Even expos ure to vessel noise at lower volumes (153 dBLeq re: 1 Pa) elicited increased plasma co rtisol concentrations in both hearing generalists and hearing sp ecialists (Wysocki et al., 2006) but white noise exposure (at 160-170 dBrms re: 1 Pa) did not lead to sustained elevated cortisol concentrations in goldfish (Smith et al., 2004). Behaviorally, Blaxter and Batty (1987) dem onstrated startle res ponses in herring when exposed to transient sound s timuli. Popper and Carlson (1 998) review se veral studies demonstrating avoidance behavior of several groups of fishes to varying sound stimuli as a

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83 method to discourage fishes from entering intakes at power plants or dams. Pearson et al. (1992) demonstrated alarm and startle responses of rockfish to acoustic stimuli emitted by geophysical survey devices (at 180peak dB re: 1 Pa), and Skalski et al. (1992) subsequently demonstrated a decreased catch-per-unit effort. White noise exposure (at 158rms dB re: 1 Pa) induced temporary hearing threshold shifts of hearing specialist fishes (A moser and Ladich, 2003), and very intense exposures (at levels ranging from 180 to 204 dB re: 1 Pa) have led to neuromast damage and loss in the inner ear of fishes ( e.g., Enger, 1981; Cox et al., 1986a, b, 1987; Ha stings et al., 1996; McCauley et al., 2003). The diversity of literature exploring effect s of anthropogenic noise on fishes renders a prediction of effects in an aquaculture setting difficult to estimate. Anthropogenic noise to which wild fishes are exposed varies greatly in sound pressure level (SPL), frequency composition, and duration of exposure. Litera ture pertaining specifi cally to effects of aquaculture noise is, on the other hand, quite sp arse, and has been reviewed in Chapter 1. I examined effects of chronic exposure to loud ambient aquarium noise on primary, secondary, and tertiary stress indices of a popular marine or namental aquarium fish, the lined seahorse ( Hippocampus erectus ). Masonjones and Babson (unpublished) demonstrated increased incidence of gas bladder disease, behavioral differences, longer gestation lengths, and fewer, smaller, and slower grow ing offspring in dwarf seahorses (H. zosterae) exposed to low frequency boat motor noise, suggesting that seahorses are also prone to effects of ambient noise. Materials and Methods Animal Accession and Husbandry Procedures Animals were collected as bycatch from shrimp trawl nets and donated by local fishermen. Upon accession, animals were quarantined for one mont h prior to transfer to the research system,

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84 receiving two treatments of chloroquine diphos phate at 10 mg/L spaced 21 days apart (for Amyloodinium spp.) and 10 minute freshwater dips (for external parasites). Animals were tagged as described in Chapter 3. Seahorses were held in same-sex groups for a minimum period of 20-21 da ys (the gestation period of H. erectus, Lourie et al., 1999) prior to experimentation. This waiting period standardizes reproductive status by allowing males to give birth and females to reach the end of th eir postmating refractory period (Masonjones and Lewis, 1996), eliminating the poten tially confounding factor of va riation in reproductive state on measures of stress. Animals were fed frozen mysids (Piscine Energetics) in the mornings and live Artemia sp. enriched with Roti-Rich in the afternoons. Tanks were siphoned clean of debris twice daily and system water changes of 10% were performed on a weekly basis. Animals received 11 hours of fluorescent light daily, with lowintensity incandescent dawn and dusk lights illuminated hour before and hour after fl uorescent lighting transition. Animals were maintained in Atlantic Ocean seawater that was diluted, chlorinated, neutralized, and pH adjusted prior to use, at 25-27C, 27-30 ppt sa linity, and at a pH of 8.0 to 8.4. Ammonianitrogen and nitrite-nitrogen le vels remained at 0 ppm thr oughout holding and experimental periods. Nitrate-nitrogen levels ranged from 2.8 to 5.6 ppm. Laboratory and Experimental Tank Design Up to fourteen 76 L glass holding aquaria held up to five animals each in same-sex groups, when not in experimentation. All exterior surfaces (except the top and front) were painted with an opaque light blue paint to standardize the vi sual environment. Each tank contained five plastic Vallisneria plants. Holding tanks were co nnected to a central filt ration system consisting of coarse mechanical prefilter pads, wet/dry biofilt ration, carbon, and ultraviolet sterilization.

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85 I employed several soundproofing design modifi cations to minimize ambient noise in the system and associated holding tanks. The wate r level in each tank was adjusted to remain approximately 2 cm above the strainer/bulkhead f itting drain assembly in the top corner of the tank, as well as above the flexible water feed tube, to minimize ambient noise that might be generated by water surface disturbance. Air st ones were placed only in the sump, not in individual tanks. The pump was soundproofed by placement within a pl astic box lined with acoustic insulation material, and an ellipsoid loop of flexible PVC pipe plumbed inline to decouple vibration traveling thr ough the rigid PVC pipe walls fr om the pump side to the tank side. The loop was supported by plastic strapping that fasten ed to the ceiling. Acoustic dampening felt strips were fitted along the corn ers and at the center of each tank bottom frame. Experimental tanks consisted of a row of sixteen 76 L glass aquaria, set-up alternately as quiet or loud tanks. Experimental tanks were pa inted, plumbed to the main system, and outfitted as above, except that bottom exterior surfaces were painted with a sand-color paint and felt strips were not applied. Each loud tank sat on a commercially available wrought iron stand. A commercially available magnetic drive inline aquarium pump (25 W, 1931 max LPH) wa s chosen as a noise stimulus circulation pump. The base of the circul ation pump was fastened to the center of a 1.9 cm thick plywood shelf that rested on the botto m frame of the stand. Each pump was outfitted with a PVC ball valve at the outpu t, and connected with hose barbs at both ends to 1.6 cm ID (internal diameter) flexible tubing that led to th e tank. Rigid PVC drain and return pipes were fastened via hose barbs to the flex ible tubing at the tank end. Re turn pipes were rotated at a 45o angle, depositing above the wa ter level (Figure 4-1).

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86 Each quiet tank sat on a recta ngular polystyrene pad (1.3 cm thick) resting on a wooden frame stand modeled after a commercially av ailable stand. The stand sat on a second polystyrene rectangular frame pad (1.9 cm thick) A smaller, commercially available magnetic driven inline aquarium pump (15W, 1022 max LPH) was chosen as a circulation pump. This pump sat on the cement floor inside the polystyre ne base frame. The pump input and output was connected via hose barbs and flexible tubing to PVC drain and return pipes as in the loud tanks, except return pipes were submerged 15 cm inside the tank, depositing below the water level. Loops of flexible tubing were in corporated into the circulation pump input and output lines, as well as the system feed line. Loops were supported by PVC collars c onnected to small bungee cords that fastened to the ceiling (for the syst em feed loops) or the wall (for the pump loops, Figure 4-2). These loops atte nuated sound vibrations trave ling inline (A. N oxon, Acoustic Sciences Corporation, pers. comm.). Tank flow rates were measured at the beginning of each trial and two weeks into each trial. Flow rates of noisy tanks were then adjusted to match the average flow rate of the quiet tanks at the time of each measurement. For behavioral observations, an opaque white curtain was hung 1 m aw ay from tank fronts to conceal observers from animals. Square holes (6.5 X 6.5 cm) were cut in the curtain in front of each tank to allow for behavior recording with a videocamera. Sound Recording and Analysis The ambient noise profile of each test tank wa s measured at the beginning and end of each of two trials. Recordings we re taken with an HTI-96-min hydr ophone (High Tech Instruments, Inc., sensitivity = -164.1 dB re: 1V/ Pa) connected to the line-in port of a laptop computer running SpectraPlus (Pioneer Hill Software), cali brated according to manufacturers instructions.

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87 One minute recordings were collected from the middle and bottom of each tank. The recording/analysis settings used in SpectraPlus are summarized in Table 2-1. Hearing generalist fishes, w ithout connections between the gas bladder and inner ear, generally have insens itive hearing above 1,000 Hz (F ay, 1988b). Hearing tests of H. erectus using the Auditory Evoked Potential (AEP) technique ( e.g., Egner and Mann, 2005) are consistent with this observation (C hapter 3). In their survey of aquaculture systems, Bart et al. (2001) also analyzed recordings in the low frequency region (25-1000 Hz), which provides a frame of reference for the sound levels to which the fish in this study were exposed. Thus, a decimation ratio of 22 was chosen in analysis se ttings, which low-pass fi ltered the signal at a cutoff of 1002 Hz. It should be noted that this reduces the total rms power measure, but the sound pressure level that is output is averaged only over the fishs ra nge of environmentally relevant hearing ability. Resulting sound f iles were post-processed by removing putative artificial electrical peaks in frequency spectra (at 60 Hz or its harmonics) using the FFT filter function in CoolEdit (Syntrilliu m Software Corporation). Animal Assignment and Preparation Tagged seahorses were randomly chosen from the holding population for each of two trials and then randomly assigned to tanks, with the restriction that each sex was evenly distributed between treatments for each trial. On Day 0, identified seahorses were anesthetized with 100 ppm of 2:1 buffered MS-222, weig hed, measured for standard le ngth (head length + trunk length + tail length, per Lourie, 2003), and placed in their assigned tanks. Ethological Methodology Data collection For each of the two trials, 1 h observations were conducted for three animals daily for 5 days, followed by one additional animal observation on the 6th day, each week for 4 weeks. This

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88 schedule enabled all 16 test an imals to be observed once per week. The weekly order of observations was kept constant for each of the 4 weeks so that each animal was observed at 7day intervals. Orders of the 3 animals observe d each day were randomized daily. Observations occurred in the afternoons, at least 1 h after morning husband ry (siphoning and feeding) but before afternoon husbandry. Observations were videotaped with a Sony Handycam Video Hi8. An ethogram was developed and programmed into the JWatcher program (Blumstein et al., 2000) to subsequently score behaviors quantitatively from videotapes. Measures and statistical analyses The following behavioral measures were chosen for analysis: The proportion of time each animal spent at the top of the tank while in sight (of the observer) The proportion of time each animal spent stationary while in sight The proportion of time the animal spent holding a holdfast over the total time the animal was stationary while in sight The number of adjustments made at rest while the animal was in sight (standardized to number of adjustments per hour while stationary for each observation session) Total number of clicks Total number of pipes Total number of gapes A repeated measures analysis of variance (ANOVA) was performed on each measure using the SAS GLM procedure (the SAS Institute, Ca ry, NC) according to the following model: y = noise + week + seahorse(noise) + noise*week + error (4-1) Of the main effects, noise contained two leve ls (loud, quiet), week contained four levels (weeks 1-4), and seahorse (nested within noise) wa s a random factor that contained 32 levels (for each animal tested). Data were not balanced, so Type III sums of squares were used. Measures

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89 containing significant interacti on effects were subsequently sorted by time and ANOVAs were run on the effect of noise at each week for each measure. I conducted a post-hoc analysis to examine hete rogeneity of variance in behavioral data. To do this, I obtained the absolute value of the difference of each individual measurement from the mean (per treatment per week) and tested these individual deviations in the above specified repeated measures ANOVA. I then log transformed original data for measures that showed significant heterogeneity of variance due to noise or the noise*week interaction and tested logtransformed data in the ANOVA model (Equati on 4-1). Reported results are from logtransformed data for which I found significant hete rogeneity of variance, and raw data for which variance was found to be homogenous. Physiological Methodology Necropsy After 30 days, animals were euthanized fo r diagnostic necropsy and physiological measurements. On the day of necropsy, no anim al husbandry was completed and activity in the room was limited to entry and exit to retrieve animals for necropsy. Human movement in front of tanks still containing animals did not occur. For retrieval, a researcher entered the room with a bucket containing a solution of 1,000 ppm of 2:1 buffered MS-222 in seawater. Anim als in each tank were quickly removed by hand and placed in the bucket. Animals were then transported to an adjacent satellite necropsy laboratory with a total of three workers present. After 1 min exposure to MS-222, the animal was immediately decapitated with a heparinized s calpel, followed by amput ation of the entire tail. One researcher obtained two glucose read ings from blood expressed by the cut surface of the head using an Ascensia Contour Glucometer (Bayer HealthCare). One researcher prepared a blood smear from blood expressed by the cut surf ace of the tail. All three researchers then

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90 collected remaining blood from the cut surfaces of the head, trunk, and tail using labeled heparinized microhematocrit t ubes. Microhematocrit tubes were sealed with clay and refrigerated until later processing. All blood was collected within 4 minutes of retrieving the animal from the experimental tank to avoid acu te stress response due to handling (cortisol concentrations rise typically rise five to ten minutes after the onset of an acute stressor in fishes, Sumpter, 1997). The animal was then reassembled, weighed, a nd measured for standard length (per Lourie, 2003). External health observations were noted, and wet mounts of skin tissue samples prepared and observed under compound microscope if grossl y visible abnormalities were present. Heads and tails were subsequently discarded. The cut ting board and animal trunk were then wiped with alcohol pads and flame-sterilized. Using sterili zed instruments and ster ile technique, the trunk wall was removed, exposing the coelomic cavity. Organs were displaced as necessary to expose the posterior kidney. One blood agar plate (TSA II 5% Sheeps Blood, Becton, Dickinson, and Company) and two Lowenstein-Jensen slants (Becton, Dickinson, and Company) were inoculated with anterior kidney cu lture samples to check for bacter ial infection. Seahorse trunks were subsequently eviscerated, organs separate d, and the condition of individual organs were noted. Individual organs were weighed, and wet mount slides of organ samples were prepared and observed under compound microscope if gros sly visual abnormalities were present. Blood processing Microhematocrit tubes were spun at 14,000 rpm for 10 minutes. Using dial calipers, the height of the red cell column wa s measured and compared to the total height of the column of whole blood to obtain packed cel l volumes (PCV). Tubes were then broken at the packed cell/plasma interface and plasma was transferred to labeled cryovials that were subsequently stored at -80oC for cortisol analysis.

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91 Blood smears were stained with Dip Quick (Jorgensen Laboratories). Smears were evaluated for leukocyte count, differential, and heterophil:lymphocyt e (H:L) ratio under microscopy at 1,000X. Each cell type (erythrocytes, lymphocyt es, monocytes, heterophils, and other granulocytes) was counted in one optical fiel d. This process was repeated for each smear until at least 2,000 cells were enumerated. Cortisol was measured using a commercially available Cortisol Enzyme Immunoassay (EIA) Kit (Cayman Chemical, Inc.). All seahor se plasma samples were extracted twice with diethyl ether prior to assay. The cortisol EIA kit for the lined seahorse was validated by the parallelism of the dilution curve of a pooled plasma sample of si x lined seahorses (not used in the experiment) with the standard curve of the kit. Based on va lidation results, a dilution factor of 1:30 was chosen for assay of experimental animals. Assays were completed according to manufacturers instru ctions and plates were read with a mi croplate reader connected to a PC with Microplate Manager software. Samples that yi elded out-of-range or questionable results were rerun in a second assay, at ei ther a 1:300 or 1:30 dilution fact or (respectively) One in-range sample from the first assay was al so run in the second assay to eval uate interassay variation. The coefficients of variation intra-assay were (mean + SE) 10.6 + 2.1% for assay 1 and 5.4 + 1.6% for assay 2. The coefficient of variation interassay was 25.6%. Bacterial culture Blood agar plates we re incubated at 25oC and checked for growth once daily for 4 d. Positive growth was subcultured and evaluated for morphology and motility, stained with a Protocol Gram Stain (Fisher Diagnostics), and then submitted to All Florida Veterinary Laboratory (Archer, FL) for identification. One of the two Lowenstein-Jensen slants per animal was incubated at 25oC and checked for growth at days 1 & 2, then once a week for 6 weeks. The other was incubated at 37oC and checked for growth daily for 6 days. Positive growth was

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92 evaluated for colony color, cell morphology, and st ained with an acid-fast Ziehl-Neelsen Stain (Becton, Dickinson, and Company). Measures & statistical analyses Measures were categorized, and in some cases transformed, as listed in Table 4-1. Among the morphological indices, the chan ge in Fulton condition factor ( K) was calculated as the difference of the Fulton condition f actor at the end of the trial minus the Fulton condition factor at the beginning of the trial. Fulton condition factor was calculated according to the following equation, after Anderson and Neumann (1996): 000,1003 L W K (4-2) Where W = weight (g) and L = length (mm). Data for categories Categories 1-5 were tested in a 3-way factorial MANOVA in SAS (The SAS Institute, Cary, NC) according to the following model: y1 y2yk = noise + sex + trial + noise*sex + noise*trial + sex*trial + noise*sex*trial + error (4-3) Of the main effects, noise contained two levels (loud, quiet), sex contai ned two levels (male, female), and trial contained two levels (1, 2). Data were not balanced, so Type III sums of squares were used. Interpretati on of MANOVA results are based on the Wilks Lambda statistic. ANOVAs of component measures within each M ANOVA were also assessed and are reported. Results focus on the main effect of noise and an y significant interaction effects involving noise. Other main effects and interactions were includ ed in the model only to partition out variability; any significant effects not pertai ning to noise are not critical to the question and are thus not reported in detail.

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93 As category 6 only included one dependent variable (incidence of disease), this measure was run as a 3-way factorial ANOVA according to th e above model. Chi-square tests were also employed to test for dependence of pathogen incidence on tank treatment. Results Sound Analysis Ambient noise in loud tanks averaged 123.3 + 1.0 dB SPL (re: 1 Pa) in the middle of the water column and 137.3 + 0.7 dB at the tank bottom. Ambi ent noise in quiet tanks averaged 110.6 + 0.6 dB in the middle of the water column and 119.8 + 0.4 dB at the tank bottom. In both positions, loud tanks were significantly louder than quiet tanks (T-test, p < 0.001, Figure 4-3). Ethological Results Ethogram I classified all observed beha viors as maintenance behavior as they occurred in the absence of any external stimulus (except for noise and surrounding habitat). No other animals, food, or observers were pres ent/visible during observati on sessions; thus, feeding and intra/interspecific interaction behaviors were neither expected nor observed. Maintenance behaviors were categorized with re gard to the animals position in relation to the tank, stationary postures, locomotion, adjustments of the tail while attached to a holdfast, and head movements (Table 4-2). Position is an arti fact of confinement and not transl atable to seahorse behavior in the wild, but in an aquarium setting, I hypothesi zed that seahorses may position themselves in quieter areas in the tank; thus, I include it here an d have subsequently quantified it (those results follow). State analyses There were no overall differences in the percen tage of time animals spent in the behavioral states that were measured. Animals in both loud and quiet tanks spent 72 + 5% (mean + SE) of

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94 observed time in the top half of the tank (F1,30 = 0.01, p = 0.914). There were no differences among weeks (F3,88 = 1.06, p = 0.372) and no treatment*w eek interaction effects (F3,88 = 1.38, p = 0.254). There was no significant heterogene ity of variance observed due to noise (F1,30 = 0.03, p = 0.855) or interaction (F3,88 = 2.14, p = 0.101) in this measure. Animals spent 93 + 2% of observed time stationary in loud tanks and 95 + 1% of observed time stationary in quiet tanks (F1,30 = 0.48, p = 0.492, Figure 4-4). Animals spent more time stationary in both treatme nts as weeks progressed (F3,88 = 3.96, p = 0.011). There were no interaction effects (F3,88 = 1.54, p = 0.211). There was no significant heterogeneity of variance observed due to noise in this measure (F1,30 = 0.89, p = 0.354), but there was a significant interaction effect (F3,88 = 4.64, p = 0.005). This is attributable to significantly gr eater variance in this measure among animals in loud tanks in week 1 (SDloud = 29.6% vs. SDquiet = 14.3%, F 1,105 = 9.07, p < 0.01). While stationary, animals spent 74 + 4% of observed time holding in loud tanks and 75 + 3% of time holding in quiet tanks (F1,31 = 0.03, p = 0.857). There were no differences among weeks (F3,86 = 1.18, p = 0.322) and no interaction effects (F3,86 = 1.80, p = 0.153). There was no significant heterogeneity of variance observed due to noise (F1,31 = 0.22, p = 0.645) or interaction (F3,86 = 0.47, p = 0.701) in this measure. Event analyses There were no overall differences in the number of adjustments made per hour of rest between treatments (37 + 6 adjustments in loud tanks and 31 + 3 adjustments in quiet tanks, F1,31 = 0.05, p = 0.825) or among weeks (F3,86 = 1.62, p = 0.191, Figure 4-5). However, there was a significant interaction effect (F3,86 = 4.14, p = 0.009). At week 1, animals in loud tanks made significantly more adjustments (69 + 18) than animals in quiet tanks (27 + 7, F1,120 = 7.40, p < 0.01). At week 2, animals in quiet tanks made significantly more adjustments (47 + 7) than

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95 animals in loud tanks (26 + 5, F1,120 = 4.70, p < 0.05). There were no differences between treatments observed at weeks 3 and 4 ( p > 0.25 for both weeks). There was significant heterogeneity of variance observed due to the interaction between noise and week (F3,86 = 4.43, p = 0.006), driven by greater variance observed in this measure among animals in loud tanks in week 1 (SDloud = 71.5 vs. SDquiet = 26.8, F 1,120 = 3.92, p < 0.01). No differences were observed between treatments (F1,30 = 0.03, p = 0.864) or among weeks (F3,86 = 0.26, p = 0.856) in the number of times animals piped during observations (Figure 4-6). There was no significant interaction effect (F3,86 = 1.93, p = 0.130). However, animals in loud tanks demonstrated significantly greater heteroge neity of variance in this measure overall (SDloud = 142.1 vs. SDquiet = 9.6, F 1,31 = 5.14, p = 0.031), and especially in week 4 (SDloud = 373.6 vs. SDquiet = 4.1, F 1,103 = 16.50, p < 0.01). No differences were observed between treatments (F1,30 = 0.45, p = 0.508) or among weeks (F3,86 = 0.93, p = 0.428) in the number of times animals clicked during observations (Figure 4-7). There was no significant interaction effect (F3,86 = 2.15, p = 0.100). However, animals in loud tanks demonstrated significantly greater heteroge neity of variance in this measure overall (SDloud = 6.7 vs. SDquiet = 3.0, F 1,31 = 5.52, p = 0.025), and especially in week 4 (SDloud = 12.3 vs. SDquiet = 1.4, F 1,110 = 23.13, p < 0.01). Animals gaped significantly more ofte n in quiet tanks (12.5 + 1.2) than in loud tanks (7.8 + 0.8, F1,31 = 7.41, p = 0.011, Figure 4-8). No significant differences were observed among weeks (F3,86 = 1.34, p = 0.267) and no significant intera ction effects were observed (F3,86 = 0.29, p = 0.835). Animals in quiet tanks al so demonstrated greater heter ogeneity of variance overall in this measure (SDloud = 6.4 vs. SDquiet = 9.3, F 1,31 = 7.41, p = 0.011).

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96Physiological Results Morphological indices Morphological indices were si gnificantly different between animals in loud tanks and animals in quiet tanks (p = 0.024). Animals in loud tanks declined in weight ( Wt = -2.22 + 0.30 g) and condition factor ( K = -0.0449 + 0.009) significantly more than did animals in quiet tanks ( Wt = -1.44 + 0.28 g, p = 0.039, K = -0.0019 + 0.0126, p = 0.007). There was no significant difference in HSI ( p = 0.812) or GSI ( p = 0.326) between tank treatments. Pertinent interaction effects were not significant. Hematological count There were no significant di fferences between treatments for hematological count measures ( p = 0.660). There were no significant effects of noise or any intera ctions pertaining to noise for any individual dependent variable. Leukocyte differential There were no significant diffe rences between treatments for leukocyte differentials as a group ( p = 0.254). While lymphocytes constituted a smaller proportion of the lymphocyte population in loud tanks than in quiet tanks (59.0 + 5.3% vs. 71.0 + 3.4%), differences between treatments were marginally significant (p = 0.051). Heterophils constituted a significantly greater proportion of the lymphocyte population in loud tanks than in quiet tanks (35.8 + 4.9% vs. 22.5 + 3.6%, p = 0.028). Consequently, the hete rophil:lymphocyte (H:L) ratio was significantly greater among animals in loud tanks (0.88 + 0.25) than animals in quiet tanks (0.36 + 0.7, p = 0.029). Pertinent interaction e ffects were insignificant.

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97Blood glucose concentration Blood glucose concentrations were not signifi cantly different between treatments (31.1 + 1.8 mg/dL in loud tanks vs. 34.0 + 2.5 mg/dL in quiet tanks, p = 0.310). There were no pertinent interaction effects. Plasma cortisol concentration Plasma cortisol concentrations were not significantly differe nt between treatments (median values of 7.23 ng/mL in loud tanks vs. 5.75 ng/mL in quiet tanks, p = 0.113). There were no pertinent interaction effects. Incidence of disease In general, animals in both treatments exhibite d variable levels of parasitism, bacterial infection, and organ pathology. Six of 16 animal s in each treatment presen ted with dermal cysts that revealed infection by Glugea heraldi as described by Blasiola (1979) and Vincent and Clifton-Hadley (1989). The digenean Dictysarca virens (described in Manter, 1947) was found in the swimbladders of 7 of 16 animals in loud tanks and 9 of 16 in quiet tanks; prevalence did not depend on treatment ( X2 test, p > 0.1). Nematodes and encysted metazoan parasites were found variably on the surfaces of organs, and in the coelom around the anus. Presence of metazoan parasites only depended on trea tment for kidneys (met azoans found on 10 of 16 animals in loud tanks and 7 of 16 animals in quiet tanks, X2 test, p < 0.05). Other gross abnormalities were commonly observed in the liver, that was enlarged, pale, vascularized, and exhibited bile accumulation in the majority of animals from both treatments. Bacteria were cultured on blood agar from three animals. Aeromonas hydrophila (Gram negative motile bacilli) were cultured from the pos terior kidneys of two males in quiet tanks. Vibrio vulnificus (Gram negative motile bacilli) was cultu red from the posterior kidney of one female in a loud tank. These results were not su fficient for statistical te sting between treatments.

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98 Lowenstein-Jensen media incubated at 37oC yielded no growth from any animals. Lowenstein-Jensen media incubated at 25 oC yielded pale yellow colo red growth of acid-fast positive bacilli by 2 weeks or thereafter (indicative of Mycobacterium sp. infection) from 7 animals in loud tanks and 6 animals in quiet ta nks; these proportions were not dependent on treatment ( X2 test, p > 0.05). The number of organs affected by pathogens were not significantly different between treatments or sexes, but trial 1 animals had fewer organs infected than trial 2 animals (2.9 + 0.4 vs. 4.1 + 0.3 organs respectively, p = 0.007). Physiological results are summarized in Table 4-3. Discussion The Stress Concept Within the paradigm of Selyes (1950) Ge neral Adaptation Syndrome (GAS), organisms initially exhibit an alarm response to a stressor (whether acute or chronic). In the presence of a persistent (i.e., chronic) stressor, the organism adjusts or compensates for the disturbance to achieve allostasis, essentially making physiologi cal compromises to ach ieve stability in a suboptimal environment (Sterling and Eyer, 1988). If the organism is unable to cope with the chronic stressor, it enters the th ird stage of the syndrome, exhaustion, that can lead to the development of a pathological condition or death. The aquaculture literature has abundant evidence of acute stressors in aquaria eliciting primary ( e.g., plasma cortisol), secondary (e.g., hematology and clinical ch emistry), and tertiary ( e.g., avoidance behavior) stress responses in fishes. Chasing ( e.g., Papoutsoglou et al., 1999), net confinement (e.g., Kubokawa et al., 1999), exposure to air (e.g., Barcellos et al., 1999; Belanger et al., 2001), handling and transport ( e.g., Iversen et al., 1998; Belange r et al., 2001,), and acute exposure to toxicants ( e.g., Jian-yu et al.,

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99 2005) have all been shown to elicit stress repon ses at these levels. The stress responses to chronic stressors encountered in aquacultu re settings, however, is less clear. In a chronic stress situation, an organism c ould be at any point along a continuum of stress response in either the second or third stage of Selyes GAS. The results of indices of stress measured in fishes exposed to chronic stressors are highly variable, and reflect points along this continuum. This picture is further confounded by variability in the nature, magnitude, and duration of the stressor, the speci es of fish tested, and even th e individuals genetic background. My results demonstrate the complexity of evalua ting chronic stress from a mosaic of primary, secondary, and tertiary response indices. Primary Stress Indices Cortisol, the primary stress response, is the i ndicator most often measured in studies of chronic stress. The common pattern that emerges in chronic stress studies of aquacultured fish, especially when cortisol is measured on a time se ries, is a peak in concentration that occurs within minutes to hours after the onset of the stressor, followed by a decline that occurs over days to weeks to resting levels that are nevertheless higher than unstressed controls. Such is the case in reported studies of chr onic confinement stress, altered photoperiods, daily chasing, and subordinate fish in a dominance hierarchy ( e.g., Pottinger et al., 2002; Biswas et al., 2004; Barcellos et al., 1999; Sloman et al., 2001; respectively). In other studies, the cortisol peak in response to the onset of a chronic stressor resolves to control levels despite the continued presence of the stressor. Crowding stress elicited this phenomenon in carp ( Cyprinus carpio, Ruane et al., 2002; Ruane and Komen, 2003), rainbow trout (Oncorhynchus mykiss) and brown trout ( Salmo trutta, Pickering and Pottinger, 1987), despite differences from controls demonstrated in the measurement of other physiological stress indices. This phenomenon of resolution of corticoi d titers to pre-stress levels in the continued

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100 presence of a stressor has been termed ideal compensation (Precht, 1958, in Schreck, 1981). Despite ideal compensation, Pickering and Pottin ger (1987) were concerned that chronically stressed fish might still be limited in their performance capacities, noting increased mortality rates in other studies, reduced growth rates in other studies as well as theirs, and reduced immunocompetence as indicated by re duced lymphocyte counts in blood of their study fish. I did not detect any significant elevations in plasma cortisol concentrations in seahorses after one month of exposure to noise. Inherent individual variability (cortisol levels ranged widely in both treatments) may have reduced sta tistical power to detect an effect, but I also believe that plasma cortisol con centrations, if they had risen at the onset of exposure, may have resolved over time via coping to achieve allostasis It would have been informative to document cortisol dynamics throughout the course of the experiment; this warrants future work. Secondary Stress Indices Of note among the secondary indices that were measured in this study is the response in leukocyte differential, that fell ju st short of demonstr ating lymphocytopenia, but did demonstrate significant heterophilia among animals in loud tanks. Cortisol induces karyorrhexis of lymphocytes (Dougherty, 1960) and increases blood heterophils but inhibits the migration of these cells to injured sites or inflammatory lesions and slows down w ound healing (Wendelaar Bonga, 1997). A suite of studies have demonstrated lymphocytopenia and/or heterophilia in response to acute and chr onic stress in fish ( e.g., McLeay, 1973; McLeay and Brown, 1974; Ellsaesser and Clem, 1986; Barcellos et al., 2004; Svobodov et al., 2006). The leukocyte differential results reported here are thus i ndicative of a chronic stress response. This pattern of change in the leukocyte differential has led scientists to invoke the heterophil to lymphocyte ratio (H:L ratio) as a measure of stress in animals. Gross and Siegel (1983) established this measure for use in birds, demonstrating that administered exogenous

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101 corticosterone (the primary stress hormone in bi rds) led to correlative increases in plasma corticosterone and H:L ratio. Since then, the H: L ratio has been used in studies of acute and chronic stress in a variety of animals such as birds ( e.g., Al-Murrani et al ., 2002; Huff et al., 2005; Campo et al., 2005, 2007), reptiles ( e.g., Case et al., 2005), and mammals ( e.g., Hansen and Damgaard, 1993; Fisher et al., 1997), including people ( e.g., Duffy et al., 2006). Among these studies, increased H:L ratios were associated with measures of primary, secondary, and tertiary stress indices, including increased plasma cortisol/corti costerone concentrations (Gross and Siegel, 1983; Hansen and Damg aard, 1993; Fisher et al., 1997), increased susceptibility to infectious disease (Al-Murrani et al., 2002; Huff et al., 2005), in creased occurrence or duration of distress behaviors (Hansen and Damgaard, 1993; Case et al., 2005; Campo et al., 2005, 2007), reduced weight, and mortality (Huff et al., 2005; Duffy et al., 2006). This study demonstrates an association between increased H:L ratios and weight loss, redu ced body condition, and increased variability in the o ccurrence of distress behavior s among animals in the loud tank group. To my knowledge, this paper is the first to employ the H:L ratio as a measure of chronic stress in fishes. Hyperglycemia is a consistent and well-docum ented response to acute stressors (Mazeaud and Mazeaud, 1981), but blood glucose responds inconsis tently in studies of chronic stress. Fish can become hypoglycemic or hyperglycemic in re sponse to various chr onic stressors (McLeay, 1973; McLeay and Brown, 1974; Pottinger et al ., 2002; Sala-Rabanal et al., 2003). In other studies, blood glucose variations in fish were not detected in response to altered photoperiod regimes, despite cortisol profile s indicative of ideal compensati on (Biswas et al., 2006) or even chronically elevated cortisol (B iswas et al., 2004). The non-remarkable results presented here are

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102 consistent with the referenced studies that al so measured blood glucose concentrations after chronic exposure to a stimulus affecting sensory systems. Acute stress typically results in hemoconcen tration and therefore increased hematocrit, mainly due to the action of catecholamines s ecreted by chromaffin cells (a primary stress response). Catecholamines cause erythrocytes to swell and also increase erythrocyte numbers by causing splenic contraction with re lease of erythrocytes into th e circulation (Wendelaar Bonga, 1997). The hematocrit response in chronic stress situ ations, on the other hand, is quite variable, showing fluctuations in either direction ( e.g., Pickering and Pottinger, 1987; Montero et al., 1999; Barcellos et al., 2004) or no response at all (McLeay, 1973; McLeay and Brown, 1974; Sala-Rabanal et al., 2003; Biswas et al., 2004). Chronic ambient noise exposure did not elicit a hematocrit response in this study. Tertiary Stress Indices Physiology Despite variations in the leukocyte differen tial between treatments, this study did not demonstrate differences in incide nce of disease between treatments. Animals in loud and quiet tanks had similar prevalence of infection by acid-fast positive bacilli, indicative of Mycobacterium sp., identical prevalence of infection by Glugea heraldi and similar prevalence of infection by the sw imbladder parasite Dictysarca virens Virtually all animals, regardless of treatment, presented with encysted internal metazo an parasites, whether they were located in the coelom or in/on organs. Results demonstrati ng greater incidence of encysted metazoans on kidneys in loud tanks may be coincidental; ofte n cysts were found on the capsules of organs and may have originated from elsewhere within th e coelom. The prevalence of pathogens in both treatments was quite surprising and speaks to the general prevalence of disease in wild specimens of this species. This variable but prevalent baseline pathogen load may have masked

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103 differences due to immunosuppression, especial ly in presence/absence data, that does not characterize or differentiate th e magnitude of pathogen load in animals between treatments. Furthermore, the time course over which animals were exposed to their treatments (one month) may not have been long enough to allow disease processes to develop differentially between treatments. Other studies (reviewed in Schreck, 1996; Ba lm, 1997; Wendelaar Bonga, 1997) have shown differential prevalen ce of infection between acutely or chronically stressed and nonstressed fish when inoculated with a specific pathogen, but this methodology was outside of the scope of the present study and could have obf uscated the causative factor of other observed stress response indicators. The hepatosomatic index (HSI) in this study wa s not affected by ambient noise. HSIs in fishes are commonly altered in response to chronic stressors that directly affect liver function, such as fasting ( e.g., Kakizawa et al., 1995), malnutrition ( e.g., Montero et al., 2001; Papoutsoglou et al., 2005), and toxicant exposure ( e.g., Datta and Kaviraj, 2003; Porter and Janz, 2003; Gagnon et al., 2006; Khan, 2006). However, the HSI can also decr ease in response to other stressors that do not intuitively affect liver function, such as crowding stress (Montero et al., 1999) and cortisol implanta tion (Vijayan and Leatherland, 1989). But Papoutsoglou et al. (2000, 2005) did not detect decreased HSIs in fishes exposed to stressful (as evidenced by chronically elevated plasma co rtisol concentrations) tank b ackground colors. Stress-inducing sensory stimuli may not have a si gnificant effect on HSI. It is worth noting, however, that many of the seahorses, regardless of treatment, pres ented with pale, enlarged, fatty livers, a common phenomenon among captive fishes (e.g., Grant et al., 1998). This may have masked any chronic stress response due to ambient noise in the measure of HSI.

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104 It is well-established that repeated acute and chronic stress can downregulate reproduction in fishes, decreasing reproductive indices, incl uding gonad size and GSI (Billard et al., 1981; Pankhurst and Van Der Kraak, 1997). Though a significant decrease in GSI was not demonstrated among seahorses housed in loud tanks a trend in this direction was nonetheless apparent among female seahorses. Seahorses demonstrated decreases in two of the arguably most important measures of success in (food) fish culture, in weight gain a nd change in condition factor. Cortisol is known to depress growth rate both direct ly and through reduced food intake ( e.g., Gregory and Wood, 1999). These mechanisms of weight/condition factor reduction could not be teased apart due to the methodology employed in this study. Howeve r, body condition results coupled with results from the leukocyte differential suggest that animal s in loud tanks may have responded with increased plasma cortisol profiles earlier in the experimental trial, with subsequent resolution under the aforementioned ideal compensation scenario It should be noted that another sensory stressor, tank color, elicited both elevated cortisol profiles and re duced growth in summer flounder (Paralichthys dentatus) (Cotter et al., 2005). In the context of the variability observed in other measures, the ability to subtract initial values from final values in these two measures eliminated a substantial degree of variability due to individual differences perhaps offering more power in the statistical tests employed for thes e measures. Van Weerd and Komen (1998) also point out the masking effect of individual differences in assessi ng chronic stress response in fishes. Behavior A trend that became apparent after reviewing quantitative behavioral data was increased variability in the loud tank group fo r several measures. Noting this I examined heterogeneity of variance more rigorously, partitioning the contribu tion of factors to this phenomenon. Orlando

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105 and Guillettes (2001) review of population responses to environmental stressors documents greater variance of responses in comparison to control populations. In fact, this phenomenon has led other workers to document s ub-populations of species with different stress-coping styles ( e.g., stress proactive or stress reactive). These populations demonstr ate bimodal distributions in physiological and behavioral stress response measur es that co-vary (Koolhaas et al., 1999; verli et al., 2007). This complex res ponse pattern within a population can lead to Type II errors if only measures of central tendency, and not dispersi on, are compared. In this section, I discuss measures of central tendency and heterogeneity of variance in behavioral results. Animals consistently display avoida nce responses to acute stressors ( e.g., Beitinger, 1990), including anthropogenic noise (e.g., Blaxter et al., 1981; Blaxter a nd Batty, 1987; Dunning et al., 1992; Knudsen et al., 1992; Knudsen et al., 1997), provided that an escape to a location where the acute stressor is no longer pres ent is available. While seahorses were confined in tanks, limiting avoidance capability, sound gradients within the tank provided the opportunity for animals to choose quieter locations. These s ound gradients were demonstrated by hydrophone recordings, in which mid-water column recordings were 9.2 dB quieter than bottom recordings in quiet tanks and 14.0 dB in loud tanks. Behavi oral results showed no differences in the percentage of time spent in the top half of the tank vs. the bottom half of the tank between treatments. However, animals in both treatments spent the majority of time in the top half of the tank, where ambient noise is quieter. This resu lt should be interpreted with caution, as other factors ( e.g., different holdfast or habitat type s) could explain this preference. Masonjones and Babson (unpublished) conducte d an experiment exposing the dwarf seahorse (Hippocampus zosterae) to boat motor noise in tanks. They found, among other measures, that H. zosterae spent less time attached to a holdfast in noise-exposed tanks than in

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106 quiet tanks. I expected to see a similar result in this study. I found similar responses, but only in week one, when animals in loud tanks made significantly more adjustments of the tail than did animals in quiet tanks. This measure was also more variable among loud tank animals. I also partitioned out a greater variability in the percentage of time spent while stationary among the loud tank animals in the first week Collectively, increased occurrence or variability in these behaviors may have indicated an irritation response by some animal s in loud tanks when attached to holdfasts emanating potentially irritating vibrations. The fact that these measures were not significantly greater (in mean or variance) am ong loud tank animals in weeks 2-4 indicate that habituation may have occurred, whereby the stim ulus was no longer perceived as irritating. A habituation response is consistent with the re sults of Dunning et al. (1992), who reported that alewives (Alosa pseudoharengus) habituated when exposed to con tinuous tones, but not to pulses of sound. It is difficult, however, to explai n why this trend reversed in the measure of adjustments in week 2, when animals in quiet tanks made significantly more adjustments. Some animals in loud tanks displa yed extremely high frequencies of clicking and piping, leading to heterogeneity of variance in these meas ures between treatments overall, and especially in week 4. Piping occurs among sick captive fishes a nd is thought to am eliorate hypoxia by making use of a thin layer of oxygen-rich water at the surface (Francis-Floyd, 1988). Piping may not serve the same function in seahorses. Seahorses extend the snout beyond the air-water interface and into the air during piping. Occasio nally seahorses expelled bubbles from the snout after piping, indicative of air in take. Piping may nonetheless consti tute a pathologic behavior. I have witnessed this behavior to occu r frequently in moribund syngnathids. Clicking in seahorses occurs in several contex ts. It is known to occur during feeding (Colson et al., 1998), and aggression/competiti on (Vincent, 1994b), though its frequency of

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107 occurrence in response to a stressor has never been tested prior to th is study. Fish (1953) noted clicking in H. erectus in response to transfers to new seaw ater containers, and in response to rapid transfer between two water bodies that differed in temperature by 12oC; these transfers may have represented acute stressors. In the context of a stressful environment, clicking might serve as a distress vocalization. Distress vocalizati ons are elicited in mammals and birds when 1) in the clutches of a predator (Hgs tedt, 1983), and 2) separated from their mothers or peers (for social animals); the latter phenomenon has been associated with a physio logical stress response and serves to advertise location to lost conspe cifics (Norcross and Newman, 1999; Stahl et al., 2002; Feltenstein et al., 2003). Mo st seahorse species (including H. erectus) are social animals, remaining pair-bonded to mates throughout at least one breeding season and perhaps longer (Foster and Vincent, 2004); a distress vocalization could serv e the latter function and be enhanced under stress. Seahorses in this expe riment were housed in isolation, providing separation stimulus, and the observed trend of increased numbers of clicks occurring among some animals in loud tanks is concordant with ph ysiological indices of in creased stress status. Further experimentation may yield interesting insights on the functi on of clicking in this and/or other social contexts. Animals in quiet tanks gaped more frequently and variably th an did animals in loud tanks. Limited observations on gaping in fishes in scientif ic literature suggests th at gaping is associated with low activity levels (Rasa, 1971), that is somewhat consistent with these results demonstrating less frequent adjustments in anim als in quiet tanks, but only in week 1. Conclusions The suite of physiological and behavioral st ress measurements examined in this study suggest that ambient noise from pumps in aquacultu re settings at the levels tested constitutes a chronic, subtle stressor to sea horses. Seahorses responded both behaviorally and physiologically

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108 with altered secondary and tertia ry stress indices indicative of a chronic stress response that has resolved to ideal compensation in an environm ent necessitating allostas is. As a result, though the primary stress index ( e.g., plasma cortisol) was not detected after one month of exposure, the chronic stressor still exerted deleterious long-te rm effects, including al tered leukocyte profiles and reduction in weight and conditi on factor. Behavioral measures suggest initial disturbance followed by habituation, until the duration of ex posure eventually elicits pathological and concomitant distress behaviors among some susceptible animals ( e.g., piping and clicking, respectively). In light of these results, seahor se aquarists and aquacult urists are advised to consider the acoustic environment of their aquaria, and to incorporate soundproofing modifications during design and set-up of facilities to avoid the debilitating consequences that chronic loud noise exposure can have on fish health, growth, and welfare.

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109 Table 4-1. Categorization of physiologi cal measures for statistical analysis. 1) Morphological Indices a) Change in weight ( wt) = End wt Beginning wt (g) b) Change in condition factor ( K) = Kend Kstart (per Anderson and Neumann, 1996) c) Hepatosomatic Index (HSI) = liver wt / (body wt gonad wt) d) Gonadosomatic Index (GSI) = gonad wt / (body wt gonad wt) 2) Hematological Count a) Leukocytes per 2,000 cells b) Lymphocytes per 2,000 cells c) Monocytes per 2,000 cells d) Heterophils per 2,000 cells e) Packed Cell Volume (%), averaged over microhematocrit tubes collected for each animal 3) Leukocyte Differential a) Lymphocytes pe r total leukocytes observed (%) b) Monocytes per total leukocytes observed (%) c) Heterophils pe r total leukocytes observed (%) d) Heterophil to Lymphocyte (H:L) ratio (square-root transformed) 4) Blood Glucose a) First reading b) Second reading 5) Plasma Cortisol: Non-parametric results were ranked and categorized as: a) First reading b) Second reading 6) Incidence of Disease: Number of tissues/organs in which pathogens were found

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110 Table 4-2. Ethogram of the lined seahorse, Hippocampus erectus: Maintenance behavior. Position Position of animal in relation to the tank. Top Any component of the animals body is loca ted in the top half of the vertical height of the tank. Bottom The entire animal is located in the bottom half of the vertical height of the tank. Stationary Postures while stationary. Perch Animal is attached to holdfast with less th an of tail. The rest of the tail, trunk, and head are extended and not in c ontact with the holdfast, and angled above the horizontal plane. Hold Animal is attached to holdfast with of tail or greater. Trunk and head are either touching or are le ss than 1cm away from holdfast. Extend Animal is attached to holdfast with le ss than of tail. Body is outstretched away from the holdfast, angled at or below the horizontal plane. Sit Animal is stationary on the bottom of the tank, with tail curled on the bottom. Head and trunk are in an upright position. Locomotion Postures while locomoting. Swim Animal travels horizontally, obliquely, or vertically through the water in an obliquely upright or upright posi tion, undulating pectoral and dorsal fins. The tail may either be curled forw ard, extended below, or extended trailing behind the animal. Skim Animal travels across the bo ttom of the tank with trunk in an obliquely upright or upright position, undulating pector al and dorsal fins. The tail drags on the bottom, outstretched behind the animal. Adjustments Movements of the tail with respect to the animals holdfast. Shift Animal undulates tail while attached to the holdfast. Head and trunk positions remain stationary in relation to the holdfast. There is no net displacement of the tail with respect to its initial position on the holdfast. Rotate Animal rotates tail and b ody around holdfast. Trunk rema ins upright during this event. Slide Animal ascends or descends a vertical holdfast by inching upward or downward through tail movements on the holdfast. Head or trunk movement is minimal. Animal may either be perched or extended, thus only the lower half of the tail is in contact with the holdfast. Head Movements Movements of the head or its parts. Click Head tilts upward instantaneous ly, hyoid protrudes, mouth opens. Associated with a click sound. Pipe Animals snout breaks water surface while opercular movements continue. Gape Animal opens mouth slowly, slightly tilts head upward, and protrudes hyoid. There is no click sound.

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111 Table 4-3. Summary of physio logical results. Significant p-values are in bold. F = MANOVA (for categories) or ANOVA (for individua l measures and organs affected), X2 = Chi-Square Test, subscripts denote de grees of freedom. NV = Not valid. Nonparametric plasma cortisol reported as 1st Quartile0.9 Dictysarca virens n = 7 n = 9 2 1 = 0.500 >0.1 Mycobacterium sp. n = 7 n = 6 2 1 = 0.130 >0.5 Organs Affected 3.75 + 0.353.31 + 0.40F1 24 = 1.02 0.322 Liver n = 10 n = 7 2 1 = 1.129 >0.1 Kidney n = 13 n = 7 2 1 = 4.800 <0.05GI Tract n = 3 n = 8 2 1 = 3.580 >0.05 Gonad n = 2 n = 5 NV NV Coelom n = 5 n = 3N V N V

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112 Figure 4-1. Experimental tank design schematic : Loud tank (see text for additional details).

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113 Figure 4-2. Experimental tank design schematic : Quiet tank (see text for additional details).

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114 Figure 4-3. Power spectra of ambien t noise in representative tanks. a = recordings from the middle of the water column, b = recordings from tank bottom. Black trac e = loud tank, gray trace = quiet tank. a b

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115 50 60 70 80 90 100 1234 Week% Time Stationary (while in sight) Figure 4-4. Proportion of time spen t stationary. Bars represent m ean values, error bars represent standard deviations. Gray bars indicate l oud tanks, white bars indi cate quiet tanks. Of type III tests of variance sorted by week, p < 0.01. 0 20 40 60 80 100 120 140 1234 WeekN Adjustments per Hour while Stationary** Figure 4-5. Number of adjustme nts made per hour while stationary. Bars represent mean values, error bars represent standard deviations. Gray bars indicate loud tanks, white bars indicate quiet tanks. Of type III tests of means sorted by week, p < 0.05, ** p < 0.01. Of type III tests of variance sorted by week, p < 0.01.

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116 0 100 200 300 400 500 1234 WeekPipes (n) Figure 4-6. Occurrence of piping. Bars represent mean values, error bars represent standard deviations. Gray bars indi cate loud tanks, white bars indi cate quiet tanks. Piping was significantly more variable among animals in loud tanks overall (type III test of variance, p = 0.031). Of type III tests of variance sorted by week, p < 0.01. 0 2 4 6 8 10 12 14 16 18 20 1234 WeekClicks (n) Figure 4-7. Occurrence of clicki ng. Bars represent mean values, error bars represent standard deviations. Gray bars indicate loud tanks, white bars indicate quiet tanks. Clicking was significantly more variable among animal s in loud tanks overall (type III test of variance, p = 0.025). Of type III tests of variance sorted by week, p < 0.01.

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117 0 5 10 15 20 25 30 1234 WeekGapes (n) Figure 4-8. Occurrence of gaping. Bars represent mean values, error bars represent standard deviations. Gray bars indi cate loud tanks, white bars indi cate quiet tanks. Overall, animals yawned significantly more often, a nd more variably, in quiet tanks than in loud tanks (type III tests of means and variance, p = 0.011).

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118 CHAPTER 5 ACOUSTIC ROLES AND EFFECTS IN PR EY CAPT URE BEHAVIOR OF LINED SEAHORSES (HIPPOCAMPUS ERECTUS) IN AQUARIA Introduction It is well known that one of the myriad effect s of stress on animals is a decrease in energy balance, as animals reallocate metabolic energy away from investment activities ( i.e., growth and reproduction) and toward homeostatic mechanisms that require further input in a stress situation ( i.e., respiration, locomotion, hydromin eral regulation, tissue repair, etc., Wendelaar-Bonga, 1997). This altered maintenance has been termed allostasis (Sterling and Eyer, 1988). There are multiple insults to energy balance during stress, including reductions in appetite, food intake, food assimilation, metabolic rate (Wendelaar-B onga, 1997), and potentially via alterations of growth promoting hormone concentrations, though the latter effect is quite va riable among fishes (Pankhurst and Van Der Kraak, 1997). Though re duced growth is a common indicator of chronic stress (Morgan and Iwama, 1997), th e contribution of each of the above-mentioned mechanisms to this effect is often unknown. In the aquaculture industry, fish growth a nd condition are important contributors to product marketability. As such, it behooves aquacultur ists to design and maintain systems and husbandry practices that minimize e xposure of fish to acute and/or chronic stressors that would negatively impact these qualities. In the design of filtration systems to optimize water quality for fish health, aquaculturists may be inadvertently exposing fish to loud ambient noise, a chronic stressor resulting in reduced growth and body condition (Chapter 4). Is this effect mediated via reduced food in take? If so, would a sound-induced reduction of intake stem from the downregulation of appetite and feeding response, or in the case of soniferous fishes that produce sound associated with feeding behavior, can loud ambient noise interfere with acoustic stunning of prey?

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119 The last question leads to clos er examination of the prey st unning hypothesis. Also termed the acoustic predation hypothesi s, it suggests that some aqua tic animals (odontocetes and snapping shrimp, in particular) stun or kill pr ey with high amplitude sound. Norris and Mhl (1983) first proposed the hypothesi s based on morphological considerations and observations in the wild. Other observations in the wild (Marten et al., 2001; Simon et al., 2005) and acoustic simulation experiments (Zagaeski, 1987; M ackay and Pegg, 1988; Martin et al., 2001) corroborate the hypothesis, but it is contentiously debated. In some cases, sounds associated with prey capture behavior cannot be dec oupled from physical disturbance caused by the behavior, rendering the cause of prey-stunning effects unknown (Simon et al., 2005). In the case of snapping shrimp, prey damage has been linke d to water jets produced by snapping claws and not to acoustic snaps caused by cavitating bubbles (Herberholz and Schmitz, 1999; Versluis et al., 2000). Furthermore, at least one acoustic simulation experiment has yielded no effects on prey behavior (Benoit-Bird et al., 2006). To the authors knowledge, the prey stunning hypothesis has not yet been proposed for any fish, but sound production is known to occur conc urrently with feeding among seahorses. The seahorse click is a broadband sound caused by the stridulation of the posterior process of the supraoccipital against the coronet and is a component of the sea horse feeding strike (Colson, 1998). Clicks can be quite loud, reachi ng peak sound pressure levels of 136.8 + 1.0 dB SPL (re: 1 Pa) and peak spectral accelerations of 1.52 X 10-3 + 1.87 X 10-4 m/s2 (Chapter 3) The seahorse thus merits consideration as a mode l for testing the prey stunning hypothesis. This study tests the prey stunning h ypothesis in the lined seahorse, Hippocampus erectus while also testing the effects of ambient noi se on prey capture ra te and success.

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120Materials and Methods Animals were accessioned, tagged, quarantined, and maintained as described in Chapter 4, except that animals received 12 hours of fluorescent light daily, with lowintensity incandescent dawn and dusk lights illuminated hour before a nd hour after fluorescent lighting transition. Throughout the experiment, temperature was maintain ed at 24C, salinity at 27 ppt, and pH at 8.3. Ammonia-nitrogen and nitrite-nitrogen levels remained at 0 ppm throughout holding and experimental periods. Nitr ate-nitrogen levels ranged from 2.8 to 5.6 ppm. Experimental Tank Design Two experimental tanks were designed as loud tanks and two as quiet tanks, as described in Chapter 4. Tank flow rates were measured on each day and averaged 439 L/h; flow rates of loud tanks were adjusted daily to match flow ra tes of quiet tanks. For behavioral observations, an opaque white curtain was hung 1 m away from ta nk fronts to conceal observers from animals. Small rectangular holes (10.5 X 7.0 cm) were cut in the curtain in front of each tank to allow for behavior recording with a videocamera. Sound Recording and Analysis The ambient noise profile of each test tank wa s measured daily. One minute recordings were taken from the middle of the water column and also at the bottom of the tank, at a sampling rate of 44.1 kbps, with an HTI-96-min hydrophone (High Tech Instruments, Inc., sensitivity = 164.1 dB re: 1V/ Pa) connected to a NOMAD Jukebox3 di gital audio recorder. The NOMAD Jukebox3 was calibrated with a 1.0Vpeak sine wave. Digital sound files (.wav) were post processe d by removing putative artificial electrical peaks in frequency spectra (at 60 Hz or its harmonics) using the FFT filter function in CoolEdit (Syntrillium Software Corporation). These noi se reduced files were then analyzed with SpectraPlus (Pioneer Hill Software), calibrated according to manufactur ers instructions.

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121 SpectraPlus analysis settings are listed in Table 2-1. From the frequency domain spectrum, peak frequency (in Hz), peak amplitude, and total RMS power (both in dBpeak re: 1 Pa) were documented. Signals were low-pass filtered at 1,002 Hz; total RMS power levels are thus summed within this frequency range, which is pertinent for hearing generalist fishes (including H. erectus Chapter 3) that have insensitive he aring above 1,000 Hz (Fay, 1988b). These measures were tested between loud and quiet tanks for both recording positions with t -tests. Muting Surgical procedure Nine animals were surgically muted after Co lson et al. (1998). Briefly, animals were anesthetized in a solution of 175 ppm of tricai ne methanesulfonate (MS-222) in tank water. The surgical site was cleaned with a diluted betadine solution prior to surgery, and sterile technique was observed. With the upper head of the anim al held above the wate r line, a longitudinal incision was made immedi ately anterior to the coronet, e xposing the posterior process of the supraoccipital. The process was clipped with rongeurs. Skin was apposed and sealed with cyanoacrylate. Eight additional animals were subjected to a control surgical procedure, where the right post-temporal process was removed us ing the same procedure (this process is not involved in sound production). All surgical wounds were treated once daily with topical application of 1% silver sulfadiazine cream for a minimum of 2 weeks or until fully healed. Click recording and analysis After healing, muting effect was checked by reco rding seahorse clicks during feeding. To do this, a polysterene fish shipping box was set up as a recording chamber within a quiet room. The box was filled with system water and the HTI-96min hydrophone was positioned in the center of the box at the middle of the water co lumn and connected to the NOMAD Jukebox3 for

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122 digital audio recording. Up to three seahorse s were placed in the box at any one time and an aliquot of live Artemia sp. was added to the box. Recording began when any one animal made a first strike and ceased when animals stopped feeding. The fish ID and timestamp of each strike were documented during recording. Documented clicks were isol ated in the center of windows of 5 s duration, copied and pasted into new, individual sound files for each click. Resulting sound files were then low-pass filtered at 1,002 Hz using CoolEdit software. Files were then calibrated for voltage in SpectraPlus according to manufacturer instructio ns and post-processed. Peak amplitude (in dBpeak re: 1 Pa) was measured in the time domain wa veform. Five to 18 clicks were measured from each animal (with a mean of 9.9 + 0.7 clicks per animal) and peak amplitudes were averaged. Average peak amplitudes were test ed between muted and control animals with a ttest. Prey Capture Experiment Each animal was tested in both a loud and a quiet tank with randomized order assignment, spaced at least 2 days apart and tested at the sa me time of day on each day. Animals were not fed on test days. Each animal was placed into a test tank and allowed to acclimate for 1 h prior to test. For each test trial, 100 liv e mysids (consisting primarily of Mysidopsis bahia ) were introduced to test tanks. A SONY Handycam 8 mm videocamera was used to film feeding behavior behind a blind for a 10 min period immediately followi ng mysid introduction. Video footage was subsequently scored usi ng JWatcher (Blumstein et al., 2007), according to the partial ethogram in Table 5-1. Tw o measures were calc ulated for analysis: % Successful Strikes (%SS) = 100 M S S SS (5-1)

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123 %SS was calculated as a measure of feedi ng proficiency. The outcomes of Unknown strikes (US) were unknown due to observational erro r and were not likely dependent upon treatment; these strikes were thus excluded from the above calculation. Total Successful Strikes (TSS) = TimeIS SSUSSS % % (5-2) TSS were calculated as a measure of feeding motivation. The construct US(%SS) was calculated to estimate the number of unknown strike s that were successful in order to include these in the total count of successful strikes. %TimeIS normalizes for variation in the amount of time an animal was in sight during filming. %SS was arcsine transformed according to Zar (1974). %SS and TSS were tested in a repeated measures ANOVA using Minitab (v. 15) according to th e following model: y = A + T + S(A) + A*T + (5-3) where y is the response variable, A is the animal treatment at two levels (muted or control), T is the tank treatment at two levels (loud or quiet), S(A) is the animal subject, nested within animal treatment, and is the error term. A and T are fixed factors, S(A) is a random factor. Results Sound Analysis Ambient noise analysis Loud tanks demonstrated a peak amplitude of 120.3 + 1.0 dBpeak SPL (re: 1 Pa, mean + SE) at a peak frequency of 55.9 + 20.0 Hz, with a tota l RMS power of 125.7 + 0.9 dBpeak at the middle of the water column. At tank bottom, loud tanks demonstrated a peak amplitude of 137.7 + 2.2 dBpeak SPL (re: 1 Pa, mean + SE) at a peak frequency of 137.8 + 15.6 Hz, with a total RMS power of 143.1 + 1.3 dBpeak. Quiet tanks demonstrated a peak amplitude of 114.3 + 0.7

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124 dBpeak SPL (re: 1 Pa, mean + SE) at a peak frequency of 7.3 + 0.6 Hz, with a total RMS power of 118.7 + 0.5 dBpeak at the middle of the water column. At tank bottom, quiet tanks demonstrated a peak amplitude of 118.2 + 1.1 dBpeak SPL (re: 1 Pa, mean + SE) at a peak frequency of 5.5 + 0.5 Hz, with a total RMS power of 124.5 + 0.6 dBpeak. Loud tanks had significantly higher peak amplitudes than quiet tanks at th e middle of the water column ( p < 0.0001) and at the tank bottom ( p < 0.0001). Loud tanks had si gnificantly higher total RMS power than quiet tanks at the middle of the water column ( p < 0.0001) and at the tank bottom ( p < 0.0001, Figure 5-1). Click analysis Clicks of control animals averaged 137.8 + 2.8 dBpeak SPL (re: 1 Pa). Clicks of muted animals averaged 125.6 + 2.4 dB. Control animals made signi ficantly louder clicks than muted animals ( p = 0.004). As the deciBel (dB) scale is a logarithmic scale, a 6 dB reduction is equivalent to a 50% reduction in sound pressure. Clicks of muted animals were thus, on average, approximately 25% as loud as cl icks of control animals. Prey Capture Control animals made 35 + 8 (Mean + SE) successful strikes with a 94 + 3% success rate in loud tanks and 26 + 5 successful strikes with a 98 + 1% success rate in quiet tanks. Muted animals made 35 + 4 successful strikes with a 96 + 1% success rate in loud tanks and 33 + 5 successful strikes with a 95 + 2% success rate in quiet tanks. There were no significant differences between muted and control animals (F1,15 = 1.07, p = 0.316), loud and quiet tanks (F1,15 = 0.67, p = 0.424), nor among individuals (F15,15 = 1.39, p = 0.267) in the percentage of successful strikes. There was no significant interaction effect of animal treatment and tank treatment (F1,15 = 0.00, p = 0.987).

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125 There were no significant differences between muted and control animals (F1,15 = 0.33, p = 0.574) nor between loud and quiet tanks (F1,15 = 1.62, p = 0.223) in the number of successful strikes made, though there were significant differen ces in the number of successful strikes made among individual animals (F15,15 = 2.48, p = 0.044). There was no significant interaction effect of animal treatment and tank treatment (F1,15 = 0.71, p = 0.411). Discussion Results of this study suggest that loud am bient noise in aquaculture settings does not suppress feeding response. Effects of noise on growth and body condition are instead likely to be mediated by physiological changes in energy ba lance required to maintain allostasis in a stressful environment (Sterling and Eyer, 1988; Wendelaar-Bonga, 1997), or perhaps via changes in expression of growth hormone (Pankhurst and Van Der Kraak, 1997). This study has also submitted the prey-st unning hypothesis to a nove l test with a novel model. The muting surgery allowed comparison of prey capture dynamics with and without its acoustic component (or with a markedly reduce d acoustic component). The reduction of sound pressure from the prey capture mechanism did not significantly reduce prey capture proficiency, discounting the prey-stunning hypothesis at least in this species a nd for this particular prey item. Because of this, the question of whether or not ambient noise interfer es with the acoustic stunning mechanism is not applicable for assessment in this model. The prey stunning hypothesis should be further explored in seahorses with different prey items. I chose live mysids in the context of th is experiment because of their rapid escape response and their widespread us e among public aquarists maintain ing syngnathids (pers. obs.). This test should be repeated with live amphipods, th at comprise the majority of the diet of wild H. erectus (Teixeira and Musick, 2001).

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126 For the seahorse, this evidence discounting an acoustic function of clicking on prey capture suggests, by the process of elimination, othe r proposed functions. While clicking prominently occurs during feeding, it also o ccurs during distress (Fish, 1953; Chapter 4), and in aggressive encounters among males competing for females (Vincent, 1994b). Clicking has not been examined in the courtship behavior of sea horses (Vincent and Sadler, 1995; Masonjones and Lewis, 1996); but in the context of seahorses sparse populati ons (Foster and Vincent, 2004), pair bonding (Vincent and Sadl er, 1995), and small home range s (Foster and Vincent, 2004); clicking, even if occurring while feeding, may si gnal an animals presence and/or location to a mate, or could advertise the pr esence of a food source to a ma te, ultimately providing for more fit offspring via increased nourishment of both pa rents. On balance, the function(s) of the seahorse click is/are still unknown and merit(s) further study. This study discounted its function in prey capture proficiency.

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127 Table 5-1. Partial ethogra m of the lined seahorse, Hippocampus erectus. Prey capture behavior Behavior Code Definition Successful strike SS Animal rapidly lifts head, depresses hyoid, opens mouth, followed by visually confirmed suction of mysid into snout Unknown strike US Animal rapidly lifts head, depresses hyoid, opens mouth, but mysid ingestion unknown. Miss M Animal rapidly lifts head, depre sses hyoid, opens mouth, followed by visually confirmed escape of mysid (suction of mysid into snout does not occur)

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128 Figure 5-1. Power spectra of ambien t noise in representative tanks. a = recordings from the middle of the water column, b = recordings from tank bottom. Black trac e = loud tank, gray trace = quiet tank. a b

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129 CHAPTER 6 SOUND, SEX, AND SEAHORSES: ACOUST I C ROLES AND EFFECTS IN COURTSHIP BEHAVIOR OF LINED SEAHORSES (HIPPOCAMPUS ERECTUS) IN AQUARIA Introduction The acoustic sense of fishes is a sensory m odality that can be overlooked in aquaculture. Fishes in aquaria are exposed to ambient nois e from many sources, such as water pumps, air bubbles, air pumps, and chiller motors, that can create a loud, cacophonous sound in a tank (Bart et al., 2001; Chapter 2). Consta nt exposure to loud noise may re present a chronic stressor, or may mask acoustic communication signals among soniferous fish es. If either (or both) mechanism(s) are at play, results may be delete rious for reproduction in captive fishes, which is a critical component of a su ccessful aquaculture operation. Manifestations of Stress in Courtship Behavior Effects of stress on measures of reproductiv e success at several levels of biological organization are documented among fishes. Co rtisol, the primary stress response hormone, downregulates the production of gonadotropins, androgens, estrogens, and vitellogenin in fishes (Carragher et al., 1989; Pankhurst and Van Der Kraak, 1997; Pickering et al., 1987). Downstream effects of cortisol and/or chroni c stress exposure include reduced oocyte diameter and gonad size, and lower surviv al and quality of progeny (Campbell et al., 1994; McCormick, 1998; Pankhurst and Van Der Kraak, 1997). But studi es examining impaired courtship behavior as a tertiary stress response in fishes are ra re. Morgan et al. (1999) tested the effects of capture/confinement stress on courts hip behavior in Atlantic cod (Gadus morhua). Stressed cod continued to display courtship behavior, but they initiated fewer courtships and were more likely to move directly to the final activity in the c ourtship sequence without pe rforming prior courtship behaviors that commonly occur in non-stressed fish.

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130 Elsewhere in the animal kingdom, the effect of stress on courtship behavior is wellfounded. Male courtship behavior is reduced or altered among male rodents exposed to chronic stress (DAquila et al., 1994; Retana-Marquez et al., 1996) and inescapable stress (Holmer et al., 2003). Psychosocial stress in sheep (Ovis aries) inhibits the ability of ewes to attract rams and the motivation of ewes to seek rams and initiate mating (Pierce et al., 2008). In a terrestrial example of sound stress, low-level military jet over-flights reduced courtship duration in harlequin ducks (Histrionicus histrionicus) for up to 1.5 h after exposure (Goudie and Jones, 2004). Among terrestrial invertebrates, thermal stress decreased courtship and mating behavior in fruit flies (Drosophila spp.; Patton and Krebs, 2001; Fasolo and Krebs, 2004). The hormones involved in the stress response and their direct effect on courtship behavior have also been investigated. Administration of corticosterone inhibits clasping in roughskin newts (Taricha granulosa) a very stereotypical courtship be havior that is easily triggered by applying pressure to the cloaca (Moore et al ., 1994; Rose and Moore, 1999; Rose, 2000). Corticotropin-releasing factor (CRF), an upstream hormone that signals the release of corticotropins ( e.g., cortisol, corticosterone) in response to stress, suppresses solicitation (a courtship behavior) in female white-crowned sparrows (Zonotrichia leucophrys; Maney and Wingfield, 1998). Cortisol also inhibits calli ng in green treefrogs (Hyla cinerea; Burmeister et al., 2001). The effect of stressors or stress hormones do not always result in a simple decrease in courtship behavior, but may elicit more comp lex alterations of the courtship and mating behavioral sequence. In some cases, stress (or exogenous corticotropin induction) increases early courtship behaviors in animals, such as mounting in rats (Retan a-Marquez et al., 1996), anogenital nosing of ewes by rams (Pierce et al., 2008), oral, tactile contac t, and dart-shooting in

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131 land snails ( Arianta arbustorum; Locher and Baur, 2002), despite reductions in later courtship behaviors or actual mating. The Functions of Clicking in Courtship Behavior of the Lined Seahorse, Hippocampus erectus The lined seahorse (Hippocampus erectus) makes a click sound, cau sed by a stridulation of the posterior process of the supraoccip ital against the coronet (Colson et al., 1998). The click has been observed in many behavioral contexts, including feeding (Colson et al., 1998), distress (Fish, 1953; Chapter 4), and in aggressive encounters among males competing for females (Vincent, 1994b). Clicking is not commonly observed in the c ontext of courtship behavior (Vincent and Sadler, 1995; Masonjones and Lewi s, 1996). But in the context of seahorses sparse populations (Foster and Vincent, 2004) pair bonding (Vincent and Sadler, 1995), and small home ranges (Foster and Vincent, 2004), clic king, even if occurri ng while feeding, may signal an animals presence and/or location to a mate, or could advertise the pres ence of a food source to a mate, ultimately providing for more fit offspring via increased nourishment of both parents. Whether intentional or incidental, clicking may communicate us eful information to a potential or current mate that may increase reproductive success. The Effects of Tank Noise on Acoustic Communication Examining the effects of loud noise exposur e on courtship behavior is confounded in species that employ acoustic comm unication in courtship behavior. In this scenario, sound may not only act as a stresso r, but it may also mask acoustic communication. Sound production is exhibited by a variety of fishes ( e.g., Fish et al., 1952), and occurs in a variety of behavioral contexts, including courtship and reproduction. Fishes use sounds pr oduced by potential mates to discriminate among species ( e.g., Delco, 1960; Gerald, 1971; Myrberg and Spires, 1972; Spanier, 1979), and to choose high quality mates ( e.g., Myrberg et al., 1986). In the context of

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132 courtship, sound production also elicits mate phonotaxis (Tavolga, 1958; Delco, 1960; Ibara et al., 1983; Myrberg et al., 1986) and intercepti on of mating by competing males (Stout, 1975; Kenyon, 1994). A loud environment in which acoustic signaling is masked may disrupt appropriate courtship and reproduc tive behaviors, potentially resulting in reduced rates of reproduction. Masking increases the hearing th reshold of fishes in an ensonified environment, requiring a signal to be louder in order to be heard. Masking noise levels pr esented in Chapter 1 are within the range of ambient noise encount ered in aquaria and aquaculture environments (Chapter 2, Bart et al., 2001). Furthermore, broadband noises cause a more pronounced masking effect than do narrowband noises (Hawkins and Chapman, 1975), t hus the broadband nature of ambient noise is of additional concern. Study Objectives The objectives of this study are thus three-fold (1) to examine the effect of chronic loud noise exposure on courtship behavior in the lined seahorse, (2) to examine the effect of sound production by mates on courtship be havior, and (3) to test the hypot hesis that loud ambient noise masks putative acoustic communication signals betw een mates, resulting in altered courtship behaviors. Materials and Methods Animal Accession, Laboratory Design, and Husbandry Procedures Animals were accessioned, tagged, quarantined, and maintained as described in Chapter 4, except that animals received 12 hours of fluorescent light daily, with lowintensity incandescent dawn and dusk lights illuminated hour before a nd after fluorescent light ing transition. Water quality was tested weekly; median results are as follows: Temperature, 26.1 C; Salinity, 29.8 ppt; pH, 8.2; NH3-N, 0.0 ppm; NO2-N, 0.0 ppm; NO3-N, 2.8 ppm.

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133 Experimental Tank Design Four 170 L aquaria were designed as experime ntal tanks. Tanks were painted on three sides with light blue paint and on the bottom with sand color paint to standardize the visual environment. Four plastic Vallisneria sp. plants were added to th e holding tanks to provide holdfasts/habitat. Two experimental tanks were designed as loud tanks and two as quiet tanks, as described in Chapter 4. Tank flow rates were measured at the introduction of each pair (monthly for each tank) and averaged 436.3 + 4.2 liters per hour. Flow rates of loud tanks were adjusted to match flow rates of quiet tanks at each measurement. For behavioral observations, an opaque white curtain was hung 1m away from tank fronts to conceal observers from animals. Small square holes (11 X 11 cm) were cut in the curtain in fr ont of each tank to allow for behavior recording with a videocamera. Sound Recording and Analysis The ambient noise profile of each test tank wa s measured at the introduction of each pair (monthly for each tank). One minute recordings we re taken from the middle of the water column and also at tank bottom, at a sampling rate of 44.1 kbps, with an HT I-96-min hydrophone (High Tech Instruments, Inc., sensitivity = -164.1 dB re: 1V/ Pa) connected to a NOMAD Jukebox3 digital audio recorder. The NOMAD Jukebox3 was calibrated with a 1.0Vpeak sine wave. Digital sound files (.wav) were post processe d by removing putative artificial electrical peaks in frequency spectra (at 60 Hz or its harmonics) using the FFT filter function in CoolEdit (Syntrillium Software Corporation). These noi se reduced files were then analyzed with SpectraPlus (Pioneer Hill Software), calibrate d according to manufactur ers instructions. SpectraPlus analysis settings are listed in Table 2-1. From the frequency domain spectrum, peak frequency (in Hz), peak amplitude, and total RM S power (the latter two measurements in dBpeak

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134 re: 1 Pa) were documented. Signals were lo w-pass filtered at 1,002 Hz; total RMS power levels are thus summed within this frequency range, which is pertinent for hearing generalist fishes (including H. erectus, Chapter 3) that have insensi tive hearing abov e 1,000 Hz (Fay, 1988b). These measures were tested between loud and quiet tanks for both recording positions with t -tests. Muting Eight males and eight females were surgic ally muted after Colson et al. (1998), as described in Chapter 5. Another 8 males and 8 females were subjected to the control surgical procedure. Courtship Experiment Experimental design & observational methods Animals were paired as size-matched male-f emale muted pairs and male-female control pairs. The courtship behavior of both types of pairs were observed in four trials in quiet tanks and in four trials in loud tanks. Each animal was used in only one trial. Animals were placed in assigned tanks on th e morning of the first day of observation before lights turned on. Pairs were observed and videotaped with a SONY 8mm Handycam for the first hour of light on each day for five days, beginning with the day of introduction. This time period was chosen because seahorses court mo st actively at dawn (Vincent, 1994b; Vincent and Sadler, 1995; Masonjones and Le wis, 1996). Observations ceased prior to day 5 if animals successfully mated (as evidenced by a full male brood pouch) or if a female dropped eggs (as evidenced by the presence of eggs at the bottom of the tank). Tanks were siphoned clean and feed introduced before lights on w ith the aid of red LED headlamps, to minimize disturbances at first light.

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135 To capture behaviors of both animals in suffi cient detail, the following rules were applied during videotaping (refer to Table 6-1 for terms). Both animals were judged as together and filmed within the frame when they were < 15 cm of each other and when at least one animal exhibited interactive behavior (usually brightening ). When an animal moved >15 cm away from the other animal, then the videographer zoomed in on only one animal. The choice of animal (male or female) to film when animals were apart alternat ed between bouts of together These videotaping rules resulted in complete covera ge of time spent together during the observation period, and partial and variable coverage of tim e spent apart for each sex during the observation period. Ethological analysis An ethogram, modified from Masonjones and Lewis (1996), Vincent (1994b), and Vincent and Sadler (1995), was constructed from observed behaviors and programmed into the JWatcher program (Blumstein et al., 2000) to subsequently score behaviors quantitatively from videotapes. Of the series of behaviors described in these works and observed by me, I sampled representative behaviors (Table 6-1) for statis tical analysis from both sexes, from across the stages of the courtship sequence, and that occurred with suffi cient frequency to permit statistical tests among treatments. While clicking is not described as a component of courts hip behavior in the published ethograms referenced, I c hose to analyze clicking behavior when not associated with feeding to further examine the hypot hesis that animals may click to present an acoustic signal to a mate. Behavioral states were measured as total time (in minutes) over th e observation period. Behavioral events were measured as counts. All behavioral stat es and most behavioral events tended to occur when animals were together; so to exclude variability in these measures due to variation in observational covera ge of single animals, occurrence of these behaviors when

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136 animals were apart were excluded from analysis. Pouch pumping occurred primarily while males were apart. To normalize for variation in videotape coverage of single animals while apart across trials, this behavior was only counted when animals were ap art, converted to a frequency measure (events per minute), and multiplied by the total time animals were apart during the observation period. Clicking also occurred primarily while animals were apart, but in order to address questions of the role of clicking in courtship behavior, clicking was counted separately for animals while together and for animals wh ile apart; the same conversion as for pouch pumping applied to clicking that occurre d while animals were apart. To compare courtship behavior among treatments and interactions, behavioral states were log transformed; behavioral even ts were square-root transformed (per Zar, 1974). Each behavior was tested in a repeated measures model with three factors using PROC GLIMMIX in SAS (The SAS Institute, 2006). Fixed factors included animal treatment (muted or control), tank treatment (quiet or loud), both class vari ables, and day (1-5), a quantita tive variable. Animals were modeled as random subjects for both G and R-side effects, and the first order auto-regressive structure was chosen to model co-variance st ructure of R-side effects. Because data transformations approximate normal distributions all data distributions were modeled as Gaussian distributions. The Kenward-Roger method was chosen to calculate denominator degrees of freedom. If results demonstrated significant interaction effects with time, two methods were followed for further analysis and pa rtitioning of effects: 1) Least-square mean differences were computed between significant treatments for each day and pvalues compared against a Bonferroni-adj usted alpha value of = 0.05/5 = 0.01 for 5 co mparisons (days 1-5), and 2) Time was modeled to examine linear (D), quadratic (D*D), cubic (D*D*D), or quartic (D*D*D*D) patterns.

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137 I also examined heterogeneity of variance in behavorial data. To do this, I obtained the absolute value of the difference of each individu al measurement from the mean of four animals per treatment combination per day, square-root transformed these individual deviations, and tested them in the repeated measures model de scribed above, following the same procedures for model parameterization and methods for furt her examination of interaction effects. Finally, to test for differences in occurrence of clicking while animals were together vs. apart, counts were averaged over days and paired ttests run between clicks that occurred while together and clicks that occurred while apart fo r both males and females, regardless of animal or tank treatment. Results Sound Analysis Loud tanks demonstrated a peak amplitude of 115.8 + 1.4 dBpeak SPL (re: 1 Pa, mean + SE) at a peak frequency of 61.5 + 7.1 Hz, with a total RMS power of 123.6 + 1.0 dBpeak at the middle of the water column. At tank bottom, loud tanks demonstrated a peak amplitude of 132.8 + 0.9 dBpeak at a peak frequency of 98.5 + 28.6 Hz, with a total RMS power of 137.6 + 0.9 dBpeak. Quiet tanks demonstrated a peak amplitude of 108.8 + 0.8 dBpeak at a peak frequency of 7.1 + 1.2 Hz, with a total RMS power of 114.7 + 0.6 dBpeak at the middle of the water column. At tank bottom, quiet tanks demonstr ated a peak amplitude of 115.9 + 1.8 dBpeak at a peak frequency of 14.6 + 6.3 Hz, with a total RMS power of 122.9 + 1.3 dBpeak. Loud tanks had significantly higher peak amplitudes than quiet tanks at th e middle of the water column ( p < 0.0001) and at the tank bottom ( p < 0.0001). Loud tanks had si gnificantly higher total RMS power than quiet tanks at the middle of the water column ( p < 0.0001) and at the tank bottom ( p < 0.0001, Figure 6-1).

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138 Ethological Analysis Tests of means Thirteen behaviors described in Table 6-1 were chosen for analysis. To maintain focus on the questions posed in this study, only results of behaviors demonstrati ng significant effects of animal treatment, tank treatment, or their interactions are presented in detail here. Results of clicking are described in full detail as its role in courtship behavior was a central question of this study. Most behaviors demonstrated either no significant effects of any fixed factor (male display, male point, female brighten, female display, female bow, female click) or demonstrated a significant effect of day only (time spent together, male pouch pump, male click, female approach) However, as elaborated below, the behavioral event of male approach demonstrated a significant effect of day (F1,25.16 = 4.39, p = 0.046) as well as an interaction effect between animal treatment and day (F1,25.16 = 7.74, p = 0.010). Female point also demonstrated a significant interaction effect betw een animal treatment and day (F1,42.2 = 5.64, p = 0.022). Significant results of Type III tests of means of fixed effects for selected behaviors are summarized in Table 6-2. In days 1-3 of the courtship sequence, males approach females with statistically similar frequencies (with mean number of approaches ranging from 1.6 + 0.7 to 4.6 + 2.5, Figure 6-2). By days 4 and 5, control males continue to a pproach females with elevated occurrence (with mean number of approaches at 4.3 + 1.1 and 6.3 + 1.2, respectively) while approach behavior declines significantly ( p < 0.003) among muted males (with mean number of approaches at 2.1 + 0.5 and 0.8 + 0.4, respectively). In days 1-3 of the courtship sequence, pointing occurs rarely among fe males, regardless of treatment (with mean number of points ranging from 0.3 + 0.3 to 0.8 + 0.6, Figure 6-3). In days 4 and 5, females of control pairs increase pointin g behavior dramatically (with mean number of

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139 points at 4.7 + 4.7 and 6.7 + 3.7, respectively) while females of muted pairs cease pointing entirely (p < 0.005). As there were no significant effects of animal treatment, tank treatment, or their interaction on clicking data from these groups were pooled for presentation (Figure 64). Male clicking demonstrated a significant effect of day (F1,36.27 = 11.11, p = 0.002), starting from 0.71 + 0.28 clicks produced in the first day and escalating to 5.16 + 1.48 clicks by day 5. In general, female clicks were produced in vary ing frequency over the 5 day period, ranging from 1.11 + 0.41 to 6.96 + 3.76 clicks. There was no significant effect of day among females (F1,26.08 = 1.61, p = 0.216). Tests of mean deviations While tests of means showed few effects of animal treatment and no effects of tank treatment, variance in many courtship behavior s differed significantly among tank treatments. There were also several signifi cant interaction effects. As heterogeneity of variance was examined as a stress response measure due to tank treatment, only significant main effects of tank treatment and significant inte raction effects involving tank tr eatment are described below. Significant type III results of mean deviations of these effects and interactions are summarized in Table 6-3. Behavioral states: There were significant differences in time trends of mean deviations of time spent together by pairs in quiet tanks and loud tanks over time (F1,25.46 = 5.07, p = 0.033), but least square means comparisons did not reveal significant differences of variance at the Bonferroni-corrected alpha value. Rather, th e pattern of change acr oss time differed between treatments; variation in time spent together fo llowed no significant time trend among animals in loud tanks (there were no signifi cant linear, quadratic, cubic, or quartic effects of day), but followed a quartic trend among animals in quiet tanks (F1,11.91 = 12.41, p = 0.004).

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140 There was significant heter ogeneity of variance in male brightening and male display between animals in loud tanks and in quiet tanks in the latter da ys of observation, demonstrated by a significant interaction effect of day and tank treatment ( male brightening, F1,19.53 = 10.61, p = 0.004, Figure 6-5; male display, F1,15.75 = 10.78, p = 0.005, Figure 6-6). Least square means comparisons revealed that males brightened a nd displayed in loud tanks for more variable durations than in quiet tanks (p < 0.007). There was no significant heterogene ity of variance due to treatments or their interactions in female brightening, but female display behavior showed significant heterogeneity of variance in the interaction of animal treatment and tank treatment (F1,31.47 = 9.38, p = 0.005); day and tank treatment (F1,22.81 = 6.90, p = 0.015); and a 3-way interaction in day, animal, and tank treatment (F1,22.81 = 37.22, p < 0.001, Figure 6-7). With regard to tank treatment, females in quiet tanks displayed for significantly more variable durations than females in loud tanks on days 3, 4, and 5 ( p < 0.002). With regard to th e interaction of animal treatme nt and tank treatment, animal treatment type was only heterogeneou s in variance in quiet tanks (F1,12.24 = 9.90, p = 0.008) and tank treatment type was only heterogene ous in variance among muted pairs (F1,11.51 = 19.31, p = 0.0001); these are due to a large standard deviation in this behavior among muted pairs in quiet tanks (at SD = 1.89), compared to a smaller rang e of standard deviati ons in other treatment groups (0.28 to 0.61). Behavioral events: There was significant heterogeneity of variance in pointing and pouch pumping between males in loud tanks and in quiet ta nks. Males in quiet tanks pointed more variably in quiet tanks th an in loud tanks overall (F1,37.91 = 12.33, p = 0.001, Figure 6-8). For both behaviors, there was a significant interaction effect of day and tank treatment ( pointing, F1,42.59 = 8.72, p = 0.005; pumping, F1,46.25 = 6.05, p = 0.018, Figure 6-9). For pointing, least

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141 square means comparisons revealed significant heterogeneity of variance in days 1 and 2 ( p < 0.003). For pumping, least square means comparisons signif icant heterogeneity of variance that differed in magnitude between quiet and loud ta nks in a variable pattern over days 2-5 ( p < 0.001). Female pointing displayed significantly gr eater variance in quiet tanks (SD=5.56) than loud tanks (SD=4.56) overall (F1,43.31 = 7.77, p = 0.008, Figure 6-10). Female clicking displayed no heterogeneity of va riance among any treatments or combinations, but male clicking displayed heterogeneity of variance over time (F1,31.07 = 41.17, p < 0.001, Figure 6-11). There was also an interaction of tank treatment and day (F1,31.07 = 4.80, p = 0.036); however, least square means comparis ons revealed no signifi cant differences of variance between tank treatments for any day wh en animal treatments are pooled. Rather, variance in male clicking followed different time trends between animals in loud and quiet tanks; there was no significant time trend among anim als in loud tanks, but variance progressed according to a quartic model among animals in quiet tanks (F1,14.08 = 6.60, p = 0.022). There was also a significant 3-way interaction (F1,31.07 = 5.43, p = 0.027). When the quartic time trend among animals in loud tanks was parsed further fo r analysis in the 3-way interaction, only the behavior of muted males demonstrated this time trend in loud tanks (F1,8.51 = 64.04, p < 0.001). Further parsing of the three-way interaction revealed least square means differences of variance at days 3,4, and 5 between control males in l oud and quiet tanks vs. muted males in quiet tanks ( p < 0.011), and at days 4 and 5 between muted ma les in loud tanks vs. muted males in quiet tanks ( p < 0.002). Occurrence of clicking Males clicked an average of 0.7 (+ 0.15) times while together and 2.6 (+ 0.6) times while apart. These differences were significant ( p = 0.007). Females clicked an average of 0.6 (+ 0.2)

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142 times while together and 3.8 (+ 1.2) times while apart. These differences were also significant ( p = 0.007). Discussion Comprehensively, these results reveal th at loud ambient tank noise does not impair courtship behavior in H. erectus via stress response. Clicking is an acoustic communication signal employed at least by males (and possibly by both sexes) in courtship behavior, and the signal elicits courtship responses by mates. However, loud ambi ent tank noise also apparently does not mask acoustic communication signals The Effect of Tank Nois e on Courtship Behavior Loud tanks had no apparent effect s on courtship behavior means by either sex. This is not entirely unexpected. Orlando and Guillettes (2001) review of popul ation responses to environmental stressors underscore s the importance of comparing va riance in response between stressed and control populations. In Chapter 4, significant behavioral results among stressed fish are dominated by changes in the variance of the beha vior as opposed to the m ean of the behavior. The hypothesis examined here is the potential fo r chronic loud noise exposure to generate a stress response that is manifested in altered cour tship behavior. It was sensible, then, to examine heterogeneity of variance in courtship behavi ors between animals in loud and quiet tanks. The predictions, then, are that animals in loud tanks should exhib it greater va riance in courtship behaviors than animals in quiet tanks. Heterogeneity of variance between animals in loud and quiet tanks was demonstrated, primarily as an interaction with day in 8 of 13 behaviors measured (but two behaviors, male and female point, demonstrated a significant main effect of tank treatment). However, in only two of these measures were variances greater in animals of loud tanks ( male brightening and male display); other measures either showed greater variance among animals in quiet tanks or variable va riance between treatments across days.

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143 Results suggest that heterogene ity of variance may arise from the distribution of data. Among these data, group variances ar e likely to be proportional to means. This was suggested by histograms created from pooled data for each m easure when these data were first explored; for almost all measures, pooled data demonstrated histograms with a high degree of kurtosis at the low end of the range and a la rge positive skew. This is intuit ive when thinking about animal behavior; animals that tend not to participate actively in courtship behavior produce behavioral data with many zeros and perhaps some low-valu es, all within a narrow distribution. Animals that participate actively in courts hip behavior, on the other hand, ma y vary widely with respect to frequency of behaviors (and/or time spent in behavioral states). The lack of apparent patterns in heterogeneity of variance across behavioral measures for tank treatment preclude sensible rationalization of these results. Based on the lack of patterning, it is not prudent to conclude from these data that loud tanks aff ect courtship behavior by means of a stress response. The Functions of Clicking in Courtship Behavior One of the observations that was readily apparent to observers without the aid of statistics was the more vigorous courtship be havior exhibited by males than by females, as is consistent with other sea horse species ( e.g., Masonjones and Lewis, 1996). In general, courtship behavior by males was robust regardless of treatment combin ation, while courtship behavior by females was comparatively rare. This general observation is reflected in the number of male courtship behaviors (four) that increased over the five da y period (as evidenced by a significant effect of day) vs. the number of female courtship behavi ors (one) that increased over time. These time trends are to be expected of the progressive courtship sequen ce as described by Masonjones and Lewis (1996).

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144 For most of the courtship behaviors examined, the effect of day was to be expected but otherwise not pertinent to this study in the absence of any interac tions. However, the effect of day in male clicking behavior is noteworthy. The increased occu rrence of clicking over time cooccurs with increased occurren ce of other documented male courtship behaviors over time and therefore suggests that c licking may be involved in the courts hip sequence. Clicking followed an upward trend over time with male seahorses, but not with female seahorses. This may suggest that if clicking is performed to communicate acoustic signals to a mate, that male to female signaling may be more prominent in the courtship sequence than female to male signaling. In this study, clicks were chosen for analysis when not associated with feeding. This stipulation was chosen to examine clicks that may be produced intentionally by a mate for the purposes of communication, as opposed to clicks that may be produced unintentionally during feeding behavior (Colson et al., 1998). The relative paucity of click beha vior when animals are together are corroborated in gobies (Tavolga, 1958); in Tavolgas study system, males called frequently when females were outside of the nest but calling ceased once females entered; other courtship and ma ting activities ensued. This may also be the case with seahorses; ac oustic communication may signal readiness to court when animals are apart and not w ithin visual range, but visual signaling prevails when animals are close. Because male clicking increases over time during the courtship behavior but while animals are apart presents some plausible hypotheses regardi ng the function of the click. In this scenario, it may serve to advertise presence and location to a mate, as well as readiness to mate. Courting males elicit phonotaxis of females by acoustic si gnaling in damselfish (Myrberg et al., 1986), gobies (Tavolga, 1958), midshipmanfish (Ibara et al., 1983), sunfish (Gerald, 1971), and toadfish

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145 (Bass, 1990). The acoustic nature of the click may also signal mate quality, as suggested by Colson et al. (1998) in seahorses and as demonstrated by Myrberg et al. (1986) for damselfishes. These results, coupled with the results of Chapter 3 demonstrating a frequency match between the peak amplitude of the click w ith the most sensitive hearing range of H. erectus, strongly suggest that the click is used in acoustic communication. So, the trend of increasing click behavior among males over the course of the courtship sequence suggests that males may be signaling ac oustically during courtship. But are females responding? This study was designed to address this question as well, and one result in particular suggests that they are. Given the rare performance of courtship behavior by females in general (as discussed earlier), the significant inte raction effect of muting over time in the female courtship behavior of pointing is quite important. Pointing is a behavior that occurs later in the courtship sequence, usually on the day of mati ng (Masonjones and Lewis, 1996). As such, an increase in female pointing behavior during the latter days of observation is expected; this was indeed the case among control animals (as ev idenced by Figure 6-3). However, among muted pairs, pointing ceased entirely in females during the latter days. Note that the latter days also correspond with increased clicking behavior among males. The removal of the males acoustic signal in the courtship repertoire by muting may thus prohibit reci procal courtship behaviors in females. Because female clicks exhibited no time tre nds, the argument for female signaling and its importance in the courtship repertoire is le ss strong, though the hypothesis for acoustic signaling while apart fits clicking patterns in females as we ll. It is noteworthy that males did not increase approach behavior over time in muted pairs (Figure 6-2), whereas this behavior progressed over time in control pairs. The courtship sequence is a complicated reciprocal signaling system that is

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146 thought to synchronize re productive states in preparation for copulation (Vincent and Sadler, 1995). A failure of muted males to escalate num ber of approaches as time progresses might suggest a downregulation of respons e due to lack of acoustic signals received by muted females, or it may reflect a lack of visual signals recei ved by females over time, perhaps because females are not receiving acoustic signals by males. Further courtship studies in which muted and nonmuted partners are paired may help to furthe r elucidate mechanisms behind these results. The Effects of Tank Noise on Acoustic Communication Though these results strongly suggest acoustic communication as a functional component of courtship behavior in Hippocampus erectus, there appears to be no masking effect of tank noise on signal reception. If masking occurred, animal treatment by tank treatment interactions would be expected. Specifically, the masking hypothesis leads to the pr ediction that courtship behaviors would be reduced among muted animals (in loud or quiet tank s) AND control animals in loud tanks, but not among control animals in quiet tanks. Only one significant animal treatment by tank treatment interaction was dete cted in the tests of mean deviations of female display behavior, but this effect wa s triggered by an unusually la rge standard deviation among displays by muted pairs in quiet tanks, whereas other treatment combinations demonstrated comparably low standard deviations. Th is pattern does not match the prediction. The lack of a masking effect is surpri sing in light of the masking phenomenon among hearing generalists and in light of a comparison of sound pressure levels of loud tanks to sound pressure levels of clicking. At around 200-300 Hz, which brackets the best hearing sensitivity of H. erectus (Chapter 3), loud tanks in this study demonstrated mean spectrum level amplitudes of 90-120 dBpeak SPL (re: 1 Pa) at tank bottom. Results from Chapter 3 demonstrate that seahorse clicks reach peak spectrum level amplitudes of 94.3 + 0.9 dBpeak SPL (re: 1 Pa). In Chapter 1, I present a range of critical ratios that must be met in order to preclude masking among hearing

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147 generalist fishes. As discussed, signals need to be from 10-60 dB (median around 20 dB) louder than background noise to be heard. In this experimental case, the spectral amplitude of clicking does not clear the mini mum critical ratio of this range. However, as discussed in Chapter 3, the clic k has several additional acoustical properties, including a sudden onset, and its broadband nature, that boost likelihood of de tection. It is also possible that seahorses are signaling while located higher in the water column, away from the louder noise profile at the tank bo ttom. Finally, as discussed in Chapter 2, seahorses may be responding to the particle acceleration component of the click as opposed to the sound pressure component. Unfortunately, lack of appropriate instrumentatio n precluded the opportunity to measure the particle acceleration of ambient noise in test tanks; but the signal-to-noise ratio may be quite different in this acoustic m odality. In general, scientific investigation of fish hearing response to the particle acceleration modality of s ound underwater is very sparse, presumably due to the lack of appropriate equi pment for measuring this modality This frustration has been expressed by others as well (Fay and Simmons, 1999; Casper et al., 2003). Future examination of this modality, for the questions pr oposed in this Dissertation, and elsewhere in the realm of fish bioacoustics, are likely to yield new insights to the sense of hearing in fishes.

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148 Table 6-1. Partial ethogram of courts hip behaviors of the lined seahorse, Hippocampus erectus. Behavior Description States Together Animals are within 15 cm of one another and at least one animal is engaged in interactive behavior ( e.g., brightening). Brighten The body of an animal blanches in colora tion, with the exception of the head and characteristic dark dorsal and ventral midlines Display An animal tucks its head downward and in, slightly off to one side, while brightened. Events Approach One animal approaches to within 15 cm of another animal Point An animal raises its head toward the water surface at an approximately 135 angle with respect to the body ax is and then lowers it to its starting position Pouch Pump Male opens brood pouch and jackknifes its tail forward rapidly, followed by a return of the tail to its starting position. Click An animal rapidly raises head, opens mouth, and depresses hyoid, with a rapid return to starting position. This behavior is often accompanied with a click sound. In this study, only clicking not associated with a food item was quantified. Bow An animal angles its body downward, orienting its lateral body surface parallel to the tank floor, often with the head pointed in the direction of a courting mate.

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149 Table 6-2. Summary of significant resu lts of Type III tests of means of fixe d effects in SAS PROC GLIMMIX. All F values have 1 degree of freedom in the numerator. den df = denominator degrees of freedom. Day (D) Animal Treatment Day (A*D) Interaction den df F p Trend den df F p Trend States Together 24.59 8.750.007Increase over time Male Brighten 14.49 10.760.005Increase over time Events Male Approach 25.16 4.390.046Increase over time 25.16 7.740.010Control > Muted @ Days 4,5 Male Pouch Pump 46.03 7.080.011Increase over time Male Click 42.39 11.110.002Increase over time Female Approach 22.64 14.170.001Increase over time Female Point 42.2 5.640.022Control > Muted @ Days 4,5

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150 Table 6-3. Summary of significant results of Type III tests of mean deviations of tank treatment effects or its interactions i n SAS PROC GLIMMIX. All F values have 1 degree of freedom in the numerator. A = animal treatment, T = tank treatment, D = day, den df = denominator degrees of freed om, NS = not significant Effect den dfF p Trend States Together T*D 25.465.070.033Loud tanks: NS time tre nd, quiet tanks: Quartic time trend Male Brighten T*D 19.5310.610.004> variance among males in loud tanks @ days 4, 5 Male Display T*D 15.7510.780.005> variance among males in loud tanks @ days 4, 5 Female Display A*T T*D A*T*D 31.47 22.81 22.81 9.38 6.90 37.22 0.005 0.015 <0.001 > variance among muted females in quiet tanks only > variance among females in quiet tanks @ days 3, 4, 5 Large variance among muted females in quiet tanks relative to other groups Events Male Point T T*D 37.91 42.59 12.33 8.72 0.001 0.005 > variance among males in quiet tanks > variance among males in quiet tanks @ days 1, 2 Male Pouch Pump T*D 46.2515.06<0.001Variable trends over days 2-5 Male Click T*D A*T*D 31.07 31.07 4.80 5.43 0.036 0.027 Loud tanks: NS time trend, quiet tanks: Quartic time trend Quartic time trend demonstrated only for muted males in loud tanks > variance among muted males in quiet tanks vs. control males in all tanks @ days 3, 4, 5 > variance among muted males in quiet tanks vs. muted males in loud tanks @ days 4, 5 Female Point T 43.317.770.008> variance among females in quiet tanks

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151 Figure 6-1. Power spectra of ambien t noise in representative tanks. a = recordings from the middle of the water column, b = recordings from tank bottom. Gray tr ace = quiet tank, black trace = loud tank. a b

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152 0 1 2 3 4 5 6 7 8 12345 DayCount* Figure 6-2. Comparisons of means (+ SE) of male approach. Data pooled for tank treatment. Gray = control pairs, black = muted pairs. Significant least-squares means differences, p < 0.003. 0 2 4 6 8 10 12 12345 DayCount* Figure 6-3. Comparisons of means (+ SE) of female point. Data pooled for tank treatment. Gray = control females, black = muted females. Significant least-squares means differences, p < 0.0005.

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153 0 2 4 6 8 10 12 12345 DayCount Figure 6-4. Comparisons of means (+ SE) of clicking between male s and females. Data pooled for tank and animal treatment. Gray = females, black = males. 0 2 4 6 8 10 12 14 16 18 12345 DaySD Time (min)* Figure 6-5. Comparisons of sta ndard deviations of brightening between males in quiet and loud tanks. Data pooled for animal treatment. Gray = quiet tanks, black = loud tanks. Significant least-squares means differences, p < 0.005.

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154 0 2 4 6 8 10 12 14 16 18 12345 DaySD Time (min)* Figure 6-6. Comparisons of st andard deviations of display between males in quiet and loud tanks. Data pooled for animal treatment. Gray = quiet tanks, black = loud tanks. Significant least-squares means differences, p < 0.007. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 12345 DaySD Time (min)* Figure 6-7. Comparisons of st andard deviations of display among females. Open circles = control females, closed circles = muted fema les, gray lines = quiet tanks, black lines = loud tanks. Significant least-squares mean differences between tank treatments ( p < 0.002).

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155 0 1 2 3 4 5 6 7 8 9 10 12345 DaySD Count** Figure 6-8. Comparisons of st andard deviations of pointing between males in loud and quiet tanks. Data pooled for animal treatment. Gray = quiet tanks, black = loud tanks. Significant least-squares means differences, p < 0.003. 0 5 10 15 20 25 30 35 40 12345 DaySD Count* Figure 6-9. Comparisons of st andard deviations of pouch pump ing between males in loud and quiet tanks. Data pooled for animal treat ment. Gray = quiet tanks, black = loud tanks. Significant least-squares means differences, p < 0.001.

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156 0 5 10 15 20 25 12345 DaySD Count Figure 6-10. Comparisons of standard deviati ons of pointing among females. Open circles = control females, closed circles = muted fema les, gray lines = quiet tanks, black lines = loud tanks. 0 2 4 6 8 10 12 14 16 12345 DaySD Count* Figure 6-11. Comparisons of standard deviati ons of clicking among males. Open circles = control males, closed circles = muted males, gray lines = quiet tanks, black lines = loud tanks. Significant least-squares means diffe rences between muted males in loud tanks vs. muted males in quiet tanks, p < 0.002.

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157 CHAPTER 7 DISCUSSION The Hearing Ability of Hippocampus erectus in the Context of Wild and Captive Acoustic Environments The lined seahorse is best described as a heari ng generalist, as discusse d in Chapter 3. As such, it has relatively insensitive hearing ab ilities (especially in comparison to hearing specialists). Sounds must therefore be relatively loud in order to be audible to the seahorse. Figure 7-1 overlays the estimated broadbound sound pressure audiogram with the range of ambient noise encountered at th e bottom of seahorse aquaria, where seahorses spend most of their lives in contact with the substrate or a holdfast resting on the substrate. Within the seahorses best hearing range (below 400 Hz), it is evident th at ambient noise profiles among many aquaria are audible to the seahorse. Compare this to Figure 7-2, which overlays the estimated broadband sound pressure audiogram with the range of noise encountered in the wild. Only the loudest sites where wild seahorses ar e collected are loud enough to be audible, and within a narrower frequency range of 100 to 300 Hz. An environment with audible ambient noise is not necessarily deleterious. To the contrary, Popper and Fay (1999) suggest that h earing in fishes evolved to eval uate the auditory scene. It may be beneficial for fishes to take advantag e of the acoustic sense to monitor the scene for sounds that might signal approachin g predators, prey, or mates, for example. But some fishes, both hearing generalists and hear ing specialists, live in natura l environments with inaudible ambient noise (Amoser and Ladich, 2005). The audible acoustic environment of aquaria to seahorses presents possible complications of masking of acoustic signals, or, alternativel y, a stress response to chronic noise exposure.

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158 Pressure vs. Displacement Sound energy is carried in two physical moda lities, as pressure waves and as particle motion. These components of sound contribute in different ways in the n ear-field and far-field of sound sources. The near-field of a sound source is dominated by local hydrodynamic flow, established by the displacement of water molecu les (particle motion) adjacent to a sphere vibrating in place (as in the cas e of a monopole sound source) or a sphere vibrating along an axis (as in the case of a dipole sound source, Bass and Clark, 2003). For a monopole sound source, the near-field predominates to a distance of: /2 (7-1) from the sound source, where = wavelength (m) and (pi) 3.14. So this distance is frequency dependent, but can be large underwater owing to the speed of sound in water, as: = c/f (7-2) where = wavelength (m), c = speed of sound (~340 m/s in air, ~1,500 m/s in water, Dusenberry, 1992), and f = frequency (Hz). For example, a 100-Hz soundwave has a near-field that propagates 2.4 m away from the sound sour ce underwater and only 0.5 m in air. Beyond this distance, the far-field becomes dominant (by / ) and is characterized by propagating waves that can generate a pressure wave in addition to particle motion. The inner ears of fishes are particle motion detectors (Popper et al ., 2003). They do not respond directly to sound pressu re. The swimbladder acts as a monopole resonator that converts sound pressure to particle motion for detection by the inner ear. In effect, the swimbladder renders some fishes sensitive to sound pressure; it also acts as an amplifier of sound (Popper et al., 2003). Some fishes have taken evolutionary advantage of this phys ical property of the swimbladder and have evolved bony or gaseous vesicular connections be tween the swimbladder and the inner ear to boost sound detection. It is generally well-accepted that hearing specialist

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159 fishes respond to both particle motion and pr essure, but are more sensitive to pressure particularly in the far-field and at frequencies above 70 Hz (Fay et al., 1982). Hearing generalist fishes, that have no specialized connections betw een the swimbladder and inner ear, have yielded equivocal data concerning the relative importance of pressure sensitivity to sound detection and processing (Cahn et al., 1968; Sand and Enger, 1973; Chapman and Johnstone, 1974; Fay and Popper, 1975; Jerk et al., 1989; Lovell et al., 2005). The same problem is true for sharks, that have no swimbladders at all (van den Berg a nd Schuijf, 1983; Myrberg, 2001). The consensus that might be drawn from this literature is that both acoustic modalities may be detected and processed by hearing generalist fishes, though th e relative contributions of each may vary with respect to distance, frequency, and amplitude. Nonetheless, the vast majority of the prim ary literature concerning hearing in fishes describes, measures, and tests sound in terms of sound pressure, even for hearing generalist fishes, that ought to be more sensitive to particle acceleration. This is pr esumably due to the lack of specialized equipment available to measure particle acceleration unde rwater; this frustration has been expressed by others (Fay and Simmons, 1999; Casper et al., 2003). I was fortunate to have access to the Acoustech Geophone for measurem ent of particle acceleration in Chapter 3. However, this unit was not available for use in any of the other studi es presented in this Dissertation. The Acoustech Geophone was an e xpensive instrument retailing for approximately $3,000 in 2006, as opposed to the pressure-sensiti ve hydrophone used in th is study that retailed for approximately $300 (D. Mann, pers. comm.; pers. obs.). The Acoustech Geophone unit has since malfunctioned and the company has gone out of business; another example of the difficulty in obtaining appropriate equipment for these measurements.

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160 The caveat to characterizing sound in terms of sound pressure only as it relates to hearing in generalists is that authors run the risk of describing a modality that animals are either not detecting and processing, or that pressure contributes relatively little information as opposed to particle acceleration, which may be the main mo dality of acoustic reception in generalists. Furthermore, the morphology of the sound waves of particle acceleration and particle motion do not necessarily correlate, especially in tank e nvironments. Due to the long wavelengths of underwater sounds at low freque ncies, a progressive sound wave is extremely difficult or impossible to produce in small tanks (Popper et al., 2003). Thus, the relationships between sound pressure and particle acceleration cannot be calculated with certa inty. Sound pressure thresholds measured in the lab among hearing generalist fishes may therefore be inadequate and misleading to describe or predict the animal s sensitivity to sound sources in its natural environment. The Function of Clicking Previous literature has reported the click to occur in several behavioral contexts, including feeding ( e.g., Colson et al., 1998) and competition for mates (Vincent et al., 1994b). Its role in courtship was previously unclear; Woods (2000) suggested a role in his observations of courtship behavior in Hippocampus abdominalis but clicking was not include d among courtship behaviors described in H. fuscus (Vincent, 1994b), H. whitei (Vincent and Sadler, 1995), or H. zosterae (Masonjones and Lewis, 1996). This dissertation elucidated the role of clicking in several behavioral c ontexts. It occurred more variably among animals in loud tanks in Ch apter 4. Coupled with physiological evidence of a chronic stress response, this suggests that the click may be a distress behavior; these results are corroborated by early observations made by Fish (1953), who noted clicking in H. erectus in response to transfers to new containers of water with different water quality parameters (perhaps

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161 representing an acute stressor). Whether or no t the distress click is of communicative value to conspecifics is unclear from the results of the Di ssertation, but it is possi ble (see Chapter 4 for a more complete discussion of this hypothesis). Because clicking occurs so ofte n and predictably in the contex t of feeding, it was prudent to evaluate any acoustic role of clicking in prey capture behavior. Howeve r, it appeared not to improve the prey capture success of H. erectus, at least when foraging on Mysidopsis bahia. The acoustic role of the click in prey capture behavi or should be further examined with other prey items (e.g., amphipods) that make up a larger component of the wild seahorses diet (Texeira and Musick, 2001). This Dissertation provides compelling evidence for the role of the click in intraspecific communication, however, especially in the contex t of courtship behavior The first line of evidence stems from Chapter 3. As previously stated, h earing in fishes is thought to have first evolved to evaluate the auditory scene (Poppe r and Fay, 1999). This theory is supported by several lines of evidence. First, most hear ing generalist fishes exhibit optimum hearing sensitivity below 500 Hz (Popper et al., 2003). This is concurrent with the observation that most shallow water ambient noise occurs in the rang e of 50 to 500 Hz (Bass and Clark, 2003). The inner ear evolved to a successful design early on in the evolutiona ry time table; it is found among the most primitive of jawless vertebrates and is ubiquitous in the vertebrate subphylum (Popper and Fay, 1999). By contrast, s ound production is found in only a subs et of fishes (Popper et al., 2003) and it has independently evolved multiple times (Bass and Clark, 2003), suggesting its origins of evolution after the evolution of the inner ear. Th e evolution of sound production for intraspecific communication is thus constrained by the limitations of the auditory system.

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162 To boost the likelihood of detection by a limited auditory sense, signals may have evolved several features. In the case of the seahorse click, sudden onset and its broadband nature boost likelihood of detection, as previously discussed. An especially compelling line of evidence is the frequency match between the peak frequency of the click (at 271 + 10 Hz for sound pressure and 265 + 22 Hz for particle acceleration) and the anim als optimum hearing sensitivity between 200 and 300 Hz (Figures 3-12 and Figure 3-13). Th is correspondence between auditory sensitivity and vocalization has been demonstrated fo r other sound-producing fishes as well ( e.g., Stabentheiner, 1988; Ladich and Yan, 1998; Ladi ch, 1999; Wysocki and Ladich, 2001; Lugli et al., 2003). The evolutionary shapin g of the signal in both the te mporal and frequency domains to accommodate the hearing ability of the animal lends strong evidence for the role of the click in intraspecific communication. The second line of evidence stems from Chapter 6. Male seahorses increased clicking behavior as the courtshi p sequence progressed over time. This correlates with other documented courtship behaviors that also in crease over time (Masonjones and Lewis, 1996; Chapter 6). This suggests that clicking may be a component behavior of the courtship sequence. The lack of discussion or examination of click behavior in courtship in other lite rature may be because clicking most often occurs while animals are apar t. This is not necessarily counter-intuitive; clicking may serve as an alternate signaling system when distance or obstr uctions prevent visual signaling. This pattern also occurs in gobi es (Tavolga, 1958); male s call frequently when females are outside of the nest but cease calli ng once females enter; other courtship and mating activities take precedence. It is also evident that animals are responding to acoustic signals in the context of courtship; male approach and female pointing did not escal ate over the course of the courtship sequence

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163 among muted pairs. This suggest s a downregulation of behavioral response in the reciprocal signaling system of the courtship repertoire (Vincent and Sadler, 1995) as a result of the removal of acoustic signals from the repertoire. Clicking may signal any one of a number of messages to a mate, including species discrimination (e.g., Delco, 1960; Gerald, 1971; Myrberg and Spires, 1972; Spanier, 1979), potential mate quality ( e.g., Myrberg et al., 1986; Colson et al., 1998), location and/or reproductive readiness (Tavolga, 1958; Delco, 1960; Ibara et al., 1983; Myrberg et al., 1986), and because clicking occurs so reliably with feed ing behavior (Colson et al., 1998), presence of a food source. Further studies in which one sex is muted while the other is not are suggested to further elucidate the functions of the click in cour tship behavior; this is especially sensible in light of observed sex differences in the trend of clicking over time during the courtship sequence. Variability in Signaling In this Dissertation, only feeding clicks were recorded and quantified, as they could be reliably produced upon the presentation of prey. It may be worthwhile to record and quantify clicking signals that occur in the absence of a food source; these clicks may be intended by the signaler as communication signals and may therefore be shaped differently in order to optimize reception by conspecifics ( e.g., they may be louder, have peak s at different frequencies, etc.). In several instances, I noted that the lined s eahorses made a very low frequency vibration that originated from within the body cavity wh en handled out of water. Occasionally, this vibration was of sufficient intensity to cause the head of the animal to vibrate visibly. This was a signal that was felt, as opposed to heard, a nd may occur in context of the animals natural history (I hypothesize that this vibration might occur during qu ivering behavior of courtship as described in Masonjones and Le wis, 1996; and Vincent, 1994b; and may be detected by the lateral line of mates). The functional significance of this signal deserves further exploration.

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164 Masking In order for biologically important signals to be heard, they must not only be audible ( i.e., above the hearing threshold of the receiving fish at a given fr equency), but they must also overcome a critical signal to noise ratio. As discussed in Chapter 2, signals must be anywhere between 10 to 60 dB (median around 20 dB) louder than the background environmental noise in order to be audible to hearing ge neralist fishes. In the case of the seahorse, mean spectrum-level amplitude of ambient noise is 82.6 + 2.9 dB SPL re: 1 Pa at 200 Hz in the wild. The putative communication signal, the click, demonstrates a mean spectrum-level peak amplitude of 94.3 + 0.9 dB at a nearby mean peak frequency of 271 + 10 Hz. This is a signal-to-noise ratio of roughly 12 dB. Other elements of the click, su ch as its sudden onset and its broadband nature, also boost its likelihood of detecti on (Hall, 1992; Yost, 2000). It mu st also be remembered that AEP methodology underestimates h earing thresholds in comparis on to behavioral methods by up to 19 dB (Hill, 2005). Together, the evidence sugge sts that clicks are likely to be audible to conspecifics, even in the background of ambient noise encountered in the wild. Testing the AEP response to click stimuli in the presence of environmentally relevant background noise would provide more conclusive evidence. By contrast, in aquarium environments, mean spectrum-level amplitude of ambient noise is 94.6 + 1.4 dB at 200 Hz at the tank bottom, which is virtually the same amplitude of the mean spectrum-level peak amplitude of the click. This would suggest that the click is much less likely to overcome the critical signal-tonoise ratio required in order to be heard by conspecifics in this artificial environment, rendering masking a li kely phenomenon occurring among seahorses in aquaria. But results of Chapter 6 do not suppor t masking as a hypothesis. Furthermore, results in Chapter 3 suggest that, in th e absence of the consideration of ambient noise, seahorse clicks are more likely to be audible in terms of particle acceleration than in terms of sound pressure

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165 (Figures 3-12 and 3-13). In light of these lines of evidence, it is suggested that animals may be responding primarily to the par ticle acceleration moda lity of sound, and not the pressure modality. In the particle acceleration modality, wh ere clicks are more likely to be audible, masking is less likely to occur. Further examinati on of the ambient noise fields in both wild and captive environments in terms of particle accele ration is needed. Also, most masking studies document sound in terms of sound pressure; maski ng studies of particle acceleration are also needed, as there is no a priori reason to expect that critical ratios would match those for sound pressure, especially in light of the lack of correlation between the two parameters in some environments. The Lateral Line The lateral line is a sensory system that was not examined in this Dissertation but may detect ambient noise and/or signals Like the inner ear, the lateral line is also an acceleration detector. But the lateral line is only stimulated by local, n ear-field, hydrodynamic flow. The lateral line is optimally sensitive at up to 70 Hz for free neuromasts or 180 Hz for canal neuromasts (Kalmijn, 1988). In the wild, ambient noise is unlikely to originate within the nearfield of the fish and is thus unlikely to be detected by the lateral line. Signals generated within close proximity of a fish ( e.g., by a mate in close courtship exchanges, by a competitor in close aggressive exhanges, or by an a pproaching predator) may be detect ed by the lateral line if they have energy below 100 or 200 Hz. In the wild, then, lateral lines may be exempt from some of the signal-to-noise ratio problems that the inner ear must resolve and may therefore be a reliable detector of low-frequency acoustic signals. In most small to medium sized aquaria, ambien t noise is likely to be generated within the near-field of the fish, and therefore low-frequency components of ambient noise are likely to be detected by the lateral line. I invite the reader to examine all the power spectra among the

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166 Figures presented in this Dissert ation. They depict sound energy in a range of 0-1000 Hz. The reader will note that in most cases, the region of loudest sound energy is below 100 Hz, precisely where lateral lines exhibit optimum sensitivity. The consequence of these observations is that masking of signals could occur with the lateral line sense (if signal amplitude is important in this frequency region); and/or the stress response may actually be mediated vi a the lateral line in a ddition to, or instead of the inner ear. In general, the lateral line has received much le ss attention than the inner ear over time. As late as 1984, Sand acknowledged the lack of experimental evidence to support the notion that the lateral line detects low-frequency sound (this is now accepted in the scientific community). Thorough investigation of a fishs e xperience of the lateral line sense would be fruitful here. In particular, I suggest comparison of lateral line detection of ambient nois e in confined vs. open environments, masking of signals by noise in the lateral line system, and the potential for chronic excitation of the lateral line system to induce ch ronic stress in fishes. The relative roles of the lateral line and inner ear in detecting communicati on signals by conspecifics is also worthy of investigation; though in the lined seahorse, the peak frequency of the click (at 271 + 10 Hz in the pressure modality and 265 + 22 Hz in the particle acceleration modality) is outside the range of sensitivity of the lateral line. The Stress Response Physiology The suite of physiological measures examined in response to chronic noise exposure in Chapter 4 suggests that seahorses present a chronic stress response. In particular, higher heteroph il to lymphocyte ratios were ob served; this is a secondary stress response representing an alteration of th e immune system. Seahor ses are already known to have a reduced immune system, characterized by the lack of gut-associated lymphoid tissue

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167 (Matsunaga and Rahman, 1998). In aquarium and aquaculture settings, syngnathids are known to be particularly susceptible to stress-induced disease ( e.g., Berzins and Greenwell, 2001, Frasca et al., 2005, Vincent, 1998). This result in lieu of these predis positions warns aquarists of the potential for loud ambient noise to render se ahorses vulnerable to opportunistic disease. Seahorses also demonstrated tertiary responses in two important meas ures in aquaculture, in weight and body condition. As the goal of aqu aculture (particularly food-fish aquaculture) is to rapidly produce fish muscle mass as the produc t of sale, these results suggest that sound stress can hinder this effort. Behavior Behavioral stress responses were more clearly elucidated when heterogeneity of variance was examined between treatments. This method is guided by abundant obs ervations and reviews by others who have documented increased varian ce in responses to stress in populations of animals ( e.g., Orlando and Guillette, 2001), perhaps due to alternative coping mechanisms employed by different subpopulations of animals ( e.g., Koolhaas et al., 1999; verli et al., 2007). Behavioral alterations exhibited in Chapter 4 we re subtle. In the first week of exposure, animals made more adjustments on holdfasts am ong loud tanks, and the number of adjustments made was more variable among an imals housed in loud tanks. The lack of differences in this measure in subsequent weeks suggests behavior al habitation to the pr esence of the noise stimulus. It wasnt until week 4 that clicking and piping, putativ e distress behaviors, began to appear more variably among animals in loud tanks. Despite a documented physiological stress re sponse, chronic loud noi se exposure did not appreciably affect feeding behavior in Chapter 5 or courtship behavi or in Chapter 6. The results of Chapter 5 are interesting in that they are counterintuitive to the physiological results of Chapter 4. Animals in loud tanks lost weight and body condition over time, but the results of

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168 Chapter 5 suggest that this may not be due to a reduced feeding response; instead, weight loss may be the result of an increased metabolic demand on the animal to maintain homeostasis in a suboptimal environment (or allostasis, Sterling and Eyer, 1988). The sum of these subtle and va riable behavioral results in spite of clear physiological results suggests that animal behavior may not al ways be a reliable indica tor of chronic stress in fishes. Solutions This dissertation documents a chronic stress response among fishes housed in loud tanks in aquaculture. The sound pressure levels that prod uced a stress response ar e well within the range of ambient noise encountered among public aquari a. In monitoring chronic stress in fishes, behavior may not always be a reliable indicator. For aquaculturists and aquarists, the non-lethal method of measuring body lengths and weights is suggested to m onitor the physiological status of fishes in culture over time as a potential indicator of chronic stress. In the design of fish-holding systems, its impor tant first to think about the natural history and the umwelt of the animal(s) intended to be kept. Does the animal spend the majority of its time swimming in the water column, or is it a be nthic-dwelling animal? Is the animal a hearing generalist or a h earing specialist? Chapter 2 demonstrates that ambient noise prof iles differ at different locations within an aquarium environment. Specifically, the ambien t noise encountered at the bottom of a tank is significantly louder than the ambient noise encoun tered in the middle of the water column. In the case of H. erectus, ambient noise at the middle of the wa ter column is not significantly louder than ambient noise in the wild, but ambient noise at the tank bottom is. Thus, benthic dwelling animals (such as H. erectus) may be more susceptible to ch ronic loud noise exposure in this region of the aquarium environment, and soundpr oofing measures should be focused on reducing

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169 noise at tank bottoms. In contrast, tank noise ma y not be loud enough to be of concern for some mid-water column dwelling fishes. Hearing specialists have greater hearing sensitiv ity, and hear at highe r frequencies, than do hearing generalists. In light of the evidence dem onstrated here that even a hearing generalist is susceptible to noise-induced stress, the acoustic environments of hearing specialists should especially be scrutinized. The dichotomy of hearing generalists vs. hearing specialists is important in the consideration of resonance in aquaria. Resonance is the tendency of a system to oscillate at maximum amplitudes at certain freq uencies, and tends to increase sound duration, frequency distribution, and amplit ude of signals (Akamatsu et al., 2002). Fortunately for hearing generalist fishes, resonant frequencies tend to fall above the hearing range in most tanks, according to the minimum resonant frequency ca lculations of Akamatsu et al. (2002). In contrast, for hearing specialists, some of whic h have sensitive hearing up to 2,000 Hz and less sensitive hearing up to 4,000 Hz (Popper et al., 2003), rectangular tanks as small as 570 L may have minimum resonant frequencies that fall into their range of best hearing sensitivity (2,000 Hz), with very small tanks (38 to 570 L) exhi biting minimum resonant frequencies in the range of 2,000 to 4,000 Hz. Tank size is thus a variable to consider, especially when housing hearing specialist fishes. Once these variables are considered, soundproof ing design modifications can be employed according to recommendations suggested by Davidson et al. (2007) and by the author in Chapter 2 of the Dissertation. These sensible recommendations promise to reduce ambient noise in aquarium and culture environments, thereby reducing chronic stress exposure among captive fishes.

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170 Figure 7-1. Comparisons of am bient noise in tank environments with broadband hearing in Hippocampus erectus. Power spectra of ambient noise recorded from the bottom of representative tanks of the seahorse s ound survey at the minimum and maximum of the range (both in solid gray ) and at the median of th e range (in solid black). Audiogram of H. erectus (adjusted to estimate aud ition of broadband noise) is represented by the dashed black line.

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171 Figure 7-2. Comparisons of ambient noise in the wild with broadband hearing in Hippocampus erectus. Power spectra of ambient noise recorded from the wild at the minimum and maximum of the range (both in solid gray) and at the median of the range (in solid black). Audiogram of H. erectus (adjusted to estimate audition of broadband noise) is represented by the dashed black line.

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190 BIOGRAPHICAL SKETCH Paul August Anderson was born and raised in Massachusetts. At an early age, he developed a love for the m arine world and b ecame practiced in the hobby of marine aquarium keeping. His fascination led him to pursue an undergraduate education in marine science at Eckerd College in St. Petersburg, FL. While there, Paul had the opportunity to explore many facets of marine science, conducting experiments and activities in nitrogen cycling in marine aquaria, marine plant physiology, marine ma mmal photo identification, and his undergraduate thesis in coral reef ecology where he examin ed and characterized a commensal relationship between territorial damselfishes and mysid school s on the reefs of Belize, Central America. After graduating college, Paul plunged back into his first love of aquaria and worked as an aquarist and educator at Mote Marine Aquarium in Sarasota, FL and subsequently as a lead aquarist and education coordinato r for The Pier Aquarium in St. Petersburg, FL. The additional skills he learned in these positions in animal care and project manageme nt prepared him to pursue graduate studies at the Univ ersity of Florida, that gave him the opportunity to explore an interesting question in the aquarium hobby and in aqu aculture from a fishs perspective: what is the umwelt of a fish in an aquarium world? Most recently, Paul has accepted the position of Conservation and Research Coordinator for The Florida Aquariums Center for Conservation in Tampa, FL. It is a thoroughly rewarding career opportunity for Paul; one in which his wide-ranging teaching and research background is employed to advance the diverse and exciting marine research and conservation initiatives of the Center.