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Description of the chemical senses of the Florida manatee, Trichechus manatus latirostris, in relation to reproduction

Permanent Link: http://ufdc.ufl.edu/UFE0043734/00001

Material Information

Title: Description of the chemical senses of the Florida manatee, Trichechus manatus latirostris, in relation to reproduction
Physical Description: 1 online resource (156 p.)
Language: english
Creator: Bills, Meghan L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: anatomy -- chemoreception -- glands -- histology -- manatee -- reproduction -- smell -- taste
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical 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 use of taste and smell is well documented in terrestrial mammals and aquatic non-mammalian species but has never been examined in fully aquatic mammals. It is thought that in mysticetes, pinnipeds, and sirenians there is olfactory detection at the water surface whereas detection within the water is mediated by taste buds. Anecdotal evidence indicates that the fully aquatic, endangered Florida manatee, Trichechus manatus latirostris, is capable of chemically sensing a female manatee in estrus. This project sought to define the chemical sensing abilities of the manatee and included a characterization of chemosensory receptive and transmission anatomy, behavioral assessment of male reaction to female manatee urine, and chemical analysis of that urine. A series of anatomic descriptions were completed including gross and microscopic examinations of the anal glands (n=11), taste buds (n=9) and olfactory epithelium (n=9) of the manatee. Each analysis included a representative sample of varying ages, sexes and causes of death. The anal glands were comparable to the apocrine intramuscular scent glands of dogs and emptied at the anorectal junction. Olfactory epithelium covers a majority of the three ethmoturbinates and a small portion of the septum. The examination of the mouth corroborated the presence of taste buds of the tongue root. The location of taste buds may allow for transport of chemicals without continuous exposure to the external environment. Immunohistochemical staining for estrogen receptor ß within the lingual root was completed on 23 animals of varying ages (10 males and 13 females). There was consistent staining within the epidermis and apparent staining within the taste buds of 18 animals. Behavioral analysis assessed a total of five adult males using urine from three different adult females. The study used a double blind, continuous sampling method in which an observer recorded the start time of individual behaviors before and after sample addition. The samples included salt or freshwater, estrous or anestrous urine. Two males exhibited a chemosensory response to at least one estrous sample from each of the three females. The urine that evoked a behavioral response was analyzed using reverse phase high performance liquid chromatography (HPLC) coupled to an Agilent time-of-flight (TOF) mass spectrometer with a positive electrospray ionization. A principle components and partial least squares-discriminant analysis of the ionic peaks showed a significant separation between 64 ions within the estrous and anestrous urine. The Florida manatee was the first fully aquatic marine mammal to provide evidence of chemical signaling both behavioral, male reaction to female urine, and anatomic, anal glands. Manatees possess potential receptors for these signals however the primary mechanism for perception of them has not yet been determined. Understanding chemical sensing in manatees provides insight into the yet undefined chemosensory abilities of other fully aquatic mammal species.
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 Meghan L Bills.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Larkin, Iske.
Local: Co-adviser: Samuelson, Don A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043734:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043734/00001

Material Information

Title: Description of the chemical senses of the Florida manatee, Trichechus manatus latirostris, in relation to reproduction
Physical Description: 1 online resource (156 p.)
Language: english
Creator: Bills, Meghan L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: anatomy -- chemoreception -- glands -- histology -- manatee -- reproduction -- smell -- taste
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical 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 use of taste and smell is well documented in terrestrial mammals and aquatic non-mammalian species but has never been examined in fully aquatic mammals. It is thought that in mysticetes, pinnipeds, and sirenians there is olfactory detection at the water surface whereas detection within the water is mediated by taste buds. Anecdotal evidence indicates that the fully aquatic, endangered Florida manatee, Trichechus manatus latirostris, is capable of chemically sensing a female manatee in estrus. This project sought to define the chemical sensing abilities of the manatee and included a characterization of chemosensory receptive and transmission anatomy, behavioral assessment of male reaction to female manatee urine, and chemical analysis of that urine. A series of anatomic descriptions were completed including gross and microscopic examinations of the anal glands (n=11), taste buds (n=9) and olfactory epithelium (n=9) of the manatee. Each analysis included a representative sample of varying ages, sexes and causes of death. The anal glands were comparable to the apocrine intramuscular scent glands of dogs and emptied at the anorectal junction. Olfactory epithelium covers a majority of the three ethmoturbinates and a small portion of the septum. The examination of the mouth corroborated the presence of taste buds of the tongue root. The location of taste buds may allow for transport of chemicals without continuous exposure to the external environment. Immunohistochemical staining for estrogen receptor ß within the lingual root was completed on 23 animals of varying ages (10 males and 13 females). There was consistent staining within the epidermis and apparent staining within the taste buds of 18 animals. Behavioral analysis assessed a total of five adult males using urine from three different adult females. The study used a double blind, continuous sampling method in which an observer recorded the start time of individual behaviors before and after sample addition. The samples included salt or freshwater, estrous or anestrous urine. Two males exhibited a chemosensory response to at least one estrous sample from each of the three females. The urine that evoked a behavioral response was analyzed using reverse phase high performance liquid chromatography (HPLC) coupled to an Agilent time-of-flight (TOF) mass spectrometer with a positive electrospray ionization. A principle components and partial least squares-discriminant analysis of the ionic peaks showed a significant separation between 64 ions within the estrous and anestrous urine. The Florida manatee was the first fully aquatic marine mammal to provide evidence of chemical signaling both behavioral, male reaction to female urine, and anatomic, anal glands. Manatees possess potential receptors for these signals however the primary mechanism for perception of them has not yet been determined. Understanding chemical sensing in manatees provides insight into the yet undefined chemosensory abilities of other fully aquatic mammal species.
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 Meghan L Bills.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Larkin, Iske.
Local: Co-adviser: Samuelson, Don A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043734:00001


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1 DESCRIPTION OF THE C HEMICAL SENSES OF TH E FLORIDA MANATEE, TRICHECHUS MANATUS L ATIROSTRIS IN RELATION TO REP RODUCTION By MEGHAN LEE BILLS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Meghan Lee Bills

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3 To my best friend and future husband, Diego Barboza: your support, patience and humor throughout this process have meant the world to me

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4 ACKNOWLEDGMENTS First I would like to thank my advisors; Dr. Isk and e Larkin and Dr. Don Samuel s on. You showed g reat confidence in me with this project and allowed me to explore an area outside of your expertise and for that I thank you. I also owe thanks to my committee members all of whom have provided valuable feedback and advice ; Dr. Roger Reep, Dr. David Powell and Dr. Bruce Schulte. Thank you to Patricia Lewis for her histological expertise. The Marine Mammal Pathobiology Laboratory staff especially Drs. Martine deWit and Chris Torno for sample collection. Thank you to Dr. Lisa Farina who observed the anal glan ds for the first time during a manatee necropsy. T hank you article from German to English Also, thanks go to Mike Sapper, Julie Sheldon, Kelly Evans, Kelly Cuthbert, Allison Gopaul, and Delphine Merle for he lp with various parts of the research. I also wish to thank Noelle Elliot for the chemical analysis Thank you to t he Aquatic Animal Health Program and specifically: Patrick Thompson and Drs. Ruth Francis Floyd, Nicole Stacy, Mike Walsh, Brian Stacy, and Jim Wellehan for their advice throughout this process To D r. Bob Bonde, Cathy Beck and everyone at USGS thank you not only for your help with my project but those of s irenian researchers everywhere I also owe much gratitude to who kept m e on track with the paperwork and requirements of a PhD student and Dr. Charles Courtney who has provided support and advice for the achievement of my career goals. Thank you very much to the facilities that allowed me to work with their captive manatees. Specifically: Marilyn Margold Parker

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5 Aquarium Joe Gaspard of Mote Marine Laboratory Finally, I would like to thank my Mom, Dad and brother for their encouragement and support not only during my PhD but for my entire college career I would also like to thank Heather Maness, Maggie Hunter, Gretchen Henry, Shannon Skevakis, Noel Takeuchi and Jen McGee for their feedback, critiques and friendship. I also could not have been successful without the love and support of my fianc, Diego Barboza. Funding was provided through the Florida Fish and Wildlife Conservation Commission, Whitney Marine Laboratory for Marine Bioscience and the University of Florida Aqua tic Animal Health Program, the University of Florida College o f Veterinary Medicine Consolidated Faculty Research Development Grant and Sigma Xi Grant in Aid of Research Research completed under US Fish and Wildlife permit #: MA067116 1 and UF IACUC Study #: 200902684.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 17 Chemorecep tion ................................ ................................ ................................ ..... 17 Olfaction ................................ ................................ ................................ ........... 17 Vomeronasal Organ ................................ ................................ ......................... 20 Taste ................................ ................................ ................................ ................ 21 Chemical Communication ................................ ................................ ....................... 25 Pheromones ................................ ................................ ................................ ..... 26 Mammalian Reproductive Chemical Communication ................................ ....... 31 Paenungulata ................................ ................................ ................................ ... 33 Aquatic Mammals ................................ ................................ ................................ ... 37 Olfaction ................................ ................................ ................................ ........... 38 Vomeronasal Organ ................................ ................................ ......................... 42 Taste ................................ ................................ ................................ ................ 43 The Florida Manatee ................................ ................................ ............................... 47 2 ANAL GLANDS OF THE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS: A POTENTIAL SOURCE OF CHEMOSENSORY SIGNALS ......... 52 Background ................................ ................................ ................................ ............. 52 Materials and Methods ................................ ................................ ............................ 54 Light Micr oscopy ................................ ................................ .............................. 55 Transmission Electron Microscopy ................................ ................................ ... 55 Results ................................ ................................ ................................ .................... 56 Light M icroscopy ................................ ................................ .............................. 58 Electron M icrosc opy ................................ ................................ ......................... 61 Discussion ................................ ................................ ................................ .............. 61 3 TASTE BUDS IN THE ORAL CAVITY OF THE FLORIDA MANATEE, WITHIN THE LINGUAL ROOT TASTE BUDS ................................ ........................ 66

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7 Background ................................ ................................ ................................ ............. 66 Ma terials and Methods ................................ ................................ ............................ 70 Gross and Microanatomy ................................ ................................ ................. 71 Taste B ud S ize and Q uantification ................................ ................................ ... 72 Immunohistochemistry ................................ ................................ ...................... 74 Transmission Electron Microscopy ................................ ................................ ... 76 Results ................................ ................................ ................................ .................... 76 ................................ ................................ ........................ 80 Ultrastructu re ................................ ................................ ................................ .... 83 Discussion ................................ ................................ ................................ .............. 84 4 THE NASAL CAVITY AND OLFACTORY EPITHELIUM OF THE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS ................................ .......... 91 Ba ckground ................................ ................................ ................................ ............. 91 Materials and Methods ................................ ................................ ............................ 94 Light Microscopy ................................ ................................ .............................. 96 Immunohistochemistry ................................ ................................ ...................... 97 Results ................................ ................................ ................................ .................... 97 Light Microscopy ................................ ................................ ............................ 100 Immunohistochemistry ................................ ................................ .................... 103 Discussion ................................ ................................ ................................ ............ 104 5 MALE FLORIDA MANAT EE, T RICHECHUS MANATUS LATRIROSTIS BEHAVIORAL RESPONSE TO FEMALE MANATEE URINE FROM DIFFERENT REPRODUCTIVE TIMEPOINTS ................................ ..................... 109 Background ................................ ................................ ................................ ........... 109 Methods ................................ ................................ ................................ ................ 112 Results ................................ ................................ ................................ .................. 117 Discussion ................................ ................................ ................................ ............ 124 6 CONCLUSIONS ................................ ................................ ................................ ... 127 APPENDIX : CHEMICAL ANALYSIS OF FEMALE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS URINE ................................ ............... 134 LIST OF REFERENCES ................................ ................................ ............................. 140 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 156

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8 LIST OF TABLES Table page 2 1 Samples used for anal gland analysis. ................................ ............................... 54 3 1 Taste bud samples collected for gross and histologica l analysis and fixed in 10% NBF ................................ ................................ ................................ ............ 71 3 2 Samples used for estrogen receptor analysis ................................ .................. 75 3 3 Taste bud and papillae number within manatee tongue roots. ........................... 78 3 4 Taste bud s ize within manatee tongue roots ................................ ...................... 79 3 5 Measured length of foliate papillae from the tongue roots of two manatees ...... 80 3 6 Amount of estrogen receptor within the epidermis of the Florida manatee tongue root. ................................ ... 82 3 7 ratings, within the taste buds of the Florida manatee. ................................ ........ 83 4 1 Samples collected for nasal cavity and olfactory epithelium evaluation .............. 95 4 2 The olfactory thickness of various manatees from different areas of the olfactory system as represented by different block numbers. ........................... 100 5 1 Male manatees assessed with female manatee urine. ................................ ..... 115 5 2 Ethogram of behavior categories performed by captive male West Indian manatees expos ed to urine of female manatees. ................................ ............ 116 A 1 Manatee urine samples used for chemical analysis. ................................ ........ 135

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9 LIST OF FIGURES Figure page 1 1 Transduction of olfactor y information during inhalation ................................ ...... 18 1 2 The transduction pathways of the VNO an d MOE within a hamster .................. 21 1 3 Transduction of signals to and within the brain during taste with food in the oral cavity.. ................................ ................................ ................................ ......... 24 2 1 Adult female (MEC0983), collecting duct (CD) approaching the anorectal junction.. ................................ ................................ ................................ ............. 56 2 2 Ventral view of a male manatee calf with an inset of the current working diagram of anal gland anatomy ................................ ................................ ......... 57 2 3 Anal glands (AG) of male fetus F MSW03160 completely ventral to the canal. 58 2 4 Collec ting duct (CD) and its contents ................................ ................................ 59 2 5 Collecting duct (CD) joining anal integument as an excretory duct .................... 59 2 6 Various histologic stai ns of adult manatee anal glands ................................ ..... 60 2 7 Smooth muscle actin stain of adult female LPZ102765 demonstrating the location of myoepithelial cells ................................ ................................ ............ 60 2 8 TEM of manatee anal glands ................................ ................................ .............. 61 3 1 Midsagittal view of manatee head ................................ ................................ ...... 72 3 2 Tongue root location and foliate papillae on side of root. Scale bars represent approximately 1 cm. ................................ ................................ ........................... 73 3 3 Taste bud measur ements. ................................ ................................ .................. 73 3 4 Typical foliate papillae with taste buds 20X and magnification of taste bud at 250X. ................................ ................................ ................................ .................. 74 3 5 Taste buds of the soft palate of LPZ102639 ................................ ...................... 77 3 6 Apparent taste buds, indicated by arrows of the nasal valve of MNW0905 ....... 77 3 7 ngual root of an adult manatees .......... 81 3 8 Electron micrographs of manatee taste buds. ................................ .................... 84

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10 3 9 Mid sagittal section of a mana tee head with a transverse section demonstrating the groove between tongue and teeth (arrow). ........................... 86 4 1 Head of a male manatee, MNW0 905, used for nasal examination ..................... 95 4 2 Section of manatee head, LPZ102765, from which olfactory and nasal samples were collected. ................................ ................................ ..................... 96 4 3 Measurement of olfactory thickness. ................................ ................................ .. 97 4 4 Manatee head with sections dividing the general area of bandsaw cuts made on a perinatal male, MEC0853. ................................ ................................ .......... 98 4 5 Cross sections of the nasal cavity of a perinatal male Florida manatee, MEC0853 ................................ ................................ ................................ ........... 99 4 6 C ross section of mucous producing cells from adult female, MNW0901, H&E 20X. ................................ ................................ ................................ .................. 100 4 7 Mucous glands of adult female, MNW0901, at approximately section 2 of Figure 4 4. ................................ ................................ ................................ ........ 101 4 8 Respiratory epithelium within the nasal cavity of the Florida manatee. ............ 101 4 9 Cross section from adult female, MNW0901, demonstr ating olfactory epithelium ................................ ................................ ................................ ......... 101 4 10 Section of manatee head along sagittal plane and corresponding epithelial lining within Ethmoturbinates II and III, and dorsal nasal turbinate (D) ............. 102 4 11 Diagram representing the turbinate system and location of epithelia l types within the manatee head ................................ ................................ .................. 103 4 12 OMP staining of olfactory epithelium from an adult female, LPZ102765 .......... 104 5 1 Urinary hormone levels of Lo relei, a female Florida manatee .......................... 113 5 2 Urinary hormone levels of Char lotte, a female Florida manatee ....................... 113 5 3 Urinary hormone levels of Sara, a female Florida manatee ............................. 114 5 4 Behavior of Snooty in 2009 in response to female urine from different reproductive time p oints ................................ ................................ .................. 119 5 5 Behavior of Lou in 2011 in response to female urine from dif ferent reproductive time points ................................ ................................ .................. 120 5 6 Behavior of Snooty in 2011 in response to female urine from diff erent reproductive time points ................................ ................................ ................... 121

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11 5 7 Behavior of Vail in 2011 in response to female urine from different reproductive time points. ................................ ................................ .................. 122 5 8 Behavior of male manatees to female urine from different reproductive time points in 2010. ................................ ................................ ................................ .. 123 6 1 Overall chemosensory receptor organs within the head of the manatee .......... 129 A 1 A PCA plot demonstrating separation between group A anestrous urine on the left and group B estrous urine on the right. ................................ ................ 137 A 2 A PLS DA analysis demonstrating separation in group A anestrous urine on top and group B estrous urine on bottom. ................................ ........................ 137

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12 LIST OF ABBREVIATION S AG Anal Glands AEC Aminoethylycarbazole BG BM Basement Membrane CD Collecting Duct CE Columnar Epithelium CM Centimeters CN Cranial Nerve COD Cause of Death CRL Crown Rump Length D Dorsal nasal turbinate E1S Estrogen FSH Follicle Stimulating Hormone G Goblet cells H&E H ematoxylin and E osin HLA Human Leucocyte Antigen HMDB Human Metabolome Database HPLC High Performance Liquid Chromatography KEGG Kyoto Encyclopedia of Genes and Genomes LH Luteinizing Hormone M Middle na sal turbinate Mi Microvilli MALDI TOF Matrix A ssisted Laser Desorption/Ionization Time of F light MHC Major Histocompatibility C omplex

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13 MMCD Madison Qingdao Metabolomics Consortium Database MOE Main Olfactory Epithelium MYO Myoepithelial cell NB Nerve Bundle NBF Neutral Buffered Formalin NG Nasal Glands NM Not Measured NST N ucleus of the S olitary T ract OE Olfactory Epithelium OMP Olfactory Marker Protein PAS Periodic A cid Schiff PCA Principle Components Analysis PCB Polychlorinate Biphenyls PG Progesterone PLS DA Partial Least Squares Discriminant Analysis QMP Queen Mandibular Protein SCC Solitary Chemosensory Cell SE Squamous Epithelium T1R Taste Receptor 1 T2R Taste Receptor 2 TEM Transmission Electron Microscopy TL Total Length TOF Time of Flight TP Taste Pore V Vein

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14 V1R Vomeronasal Receptor 1 V2R Vomeronasal Receptor 2 VNO Vomeronasal Organ m micrometer

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Doctor of Philosophy DESCRIPTION OF THE C HEMICAL SENSES OF TH E FLORIDA MANATEE, TRICHECHUS MANATUS L ATIROSTRIS IN RELATION TO REP RODUCTION By Meghan Lee Bills December 2011 Chair: Iskande V. Larkin Cochair: Don A. Samuelson Major: Veterinary Medical Sciences The use of taste and smell is well documented in terrestrial mammals and aquatic non mammalian species but has never been examined in fully aquatic mammals. It is thought that in mysticetes, pinnipeds, and sirenians there is ol factory detection at the water surface whereas detection within the water is mediated by taste buds. Anecdotal evidence indicates that the fully aquatic, endangered Florida manatee, Trichechus manatus latirostris is capable of chemically sensing a female manatee in estrus. This project sought to define the chemical sensing abilities of the manatee and included a characterization of chemosensory receptive and transmission anatomy behavioral assessment of male reaction to female manatee urine and chemical analysis of that urine A series of anatomic descriptions were completed including gross and microscopic examinations of the anal glands (n=11) taste buds (n=9) and olfactory epithelium (n=9) of the manatee. Each analysis included a representative sample of varying ages, sexes and causes of death. The anal glands were comparable to the apocrine intramuscular scent glands of dogs and emptied at the anorectal junction. Olfactory epithelium covers

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16 a majority of the three ethmoturbinates and a small portion of the septum. The examination of the mouth corroborated the presence of taste buds of the tongue root The location of taste buds may allow for transport of chemicals without continuous exposure to the external environment. Immunohistochemical staining for estrogen 10 males and 13 females). There was consistent staining within the epidermis and apparent staining within the taste buds of 18 animals. Behavioral analysis assessed a total of five adult males using urine from three different adult females. The study used a double blind, continuous sampling method in which an observer recor ded the start time of individual behavior s before and after sample addition. The samples included salt or freshwater, estr o us or anestr o us urine. Two males exhibited a chemosensory response to at least one estr o us sample from each of the three femal es. The urine that evoked a behavioral response was analyzed using reverse phase high performance liquid chr omatography (HPLC) coupled to an Agilent time of flight (TOF) mass spectrometer with a positive electrospray ionization A principle components and p artial l east s quares discriminant a nalysis of the ionic peaks showed a significant separation between 64 io ns within the estr o us and anestr o us urine. The Florida manatee was the first fully aquatic marine mammal to provide evidence of chemical signaling both behavioral, male reaction to female urine, and anatomic, anal glands. Manatees possess potential receptors for these signals however t he primary mechanism for perception of them has not yet been determined. Understandin g chemical sensing in manatees provide s insight into the yet undefined chemosensory abilities of other fully aquatic mammal species.

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17 CHAPTER 1 INTRODUCTION Chemoreception Within the field of sensory systems, t he area of chemoreception has had limited study and a majority of current research has focused on laboratory species A ll animals studied have developed the ability to discriminate between small variations in chemical structure and the signal blends used for communication can be extremely complex. C hemoreception varies from othe r senses because unlike vision or hearing the subject cannot scan the environment but must come into direct contact with the chemicals. For these reasons, the senses of taste and smell are more difficult to investigate compared to the other senses and ther e are many aspects of the interaction s between chemicals and their receptors that are not well understood. However, the use of chemical signals is ubiquitous among aquatic and terrestrial animals and vital to locating food, evading predators and reproducin g successful ly Olfaction Of the sensory systems, odors are one of the most complex forms of stimuli to delineate and there are still many facets of the ligand receptor bond that are not yet understood. Not only is the chemical signal itself important in the processing of olfactory information but also whether in air or water, is equally important ( Finger et al 200 0 ). The relaying of olfactory information to the brain begins with heptahelical G protein coupled receptors in the peripheral processes of bipolar neurons in the nasal cavity The centrally directed processes of these neurons collect as the olfactory ner ve filaments of cranial nerve I and terminate in the olfactory bulb ( Ache and Zhainazarov

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18 1995 ) ( Figure 1 1) Genetic data indicate that there are 400 1000 different olfactory receptors in mammals (Parmentier et al 1992, Ressler et al. 1994) whereas only 50 100 appear to exist in fish (Ngai et al. 1993, Barth et al. 1996). Figure 1 1. Transduction of olfactory information during inhalation. Air flows indicated by dashed and dotted lines AM, amygdala; LOFC, lateral orbitofrontal cortex; MOFC, medial or bitofrontal cortex; OB, olfactory bulb; OC, olfactory co rtex; OE, olfactory epithelium. Adapted from Shepherd 2006. Each olfactory receptor is encoded for by single exon genes which are distributed in clusters across multiple chromosomes (Finger et al. 2000). There are currently two known receptor classes termed type I and type II which evolved from common ancestral genes. The absence of class I receptors in terrestrial mammals and birds and the ir existence in fish indicates that these receptors are re sponsible for

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19 detection of water soluble odorants. Type II receptors are thought to be specialized for volatile odorant detection. This is supported by the presence of nonfunctional pseudogenes of type II receptors which are found in the genome of the str iped dolphin, Stenella coeruleoalba This correlates with the loss of olfaction as ancestral dolphins entered an aquatic environment. The absence of t ype I receptors indicates that o dontocetes do not have the ability to perceive aquatic odorants (Freitag e t al. 1998). These conclusions have been supported by numerous publications ( Marchand et al. 2004 Levasseur et al. 2007) and although more research is needed on this topic the categorization of type I and II receptor classes is the current working model The differences in the amount and type of receptors between aquatic and terrestrial species indicates an adaptation to environment because aquatic animals encounter a smaller number of water soluble molecules compared to the variety of hydrophobic, volat ile compounds available to terrestrial animals. For both type I and II receptors, m any non neuronal factors can affect how a signal is coded and subsequent processing of information in the brain. For example, once an odor has reached the mucus or sensilar fluid of the nasal cavity it can be binding proteins, or it can be cleared by odorant degrading proteins. A receptor can only detect one type of signal but any given signal may bind to many receptors. In addition, once a receptor has been bound by an odorant it cannot send another action potential until that odorant has been removed and this process may require an odorant degrading molecule ( Finger et al 2000 ). After the signal from the main olfactory epithelium (MOE ) has synapsed on the olfactory bulb the second synapse occurs on one of many higher olfactory centers

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20 located in the cortex. Olfactory signals most commonly project to the piriform cortex. The limbic system has an olfactory component that includes portio ns of the piriform cortex, amygdala, and hippocampus ( Finger et al. 2000 ). Comparative s tudies have correlated decline in olfactory use and olfactory bulb size within a species with a decrease in the size of the limbic system (Reep et al. 2007). Higher olfactory centers then project to subcortical and neocortical areas or can cycle back to the olfactory epithelium. The direct link from olfactory receptors to the olfactory bulb allows for almost immediate response to stimuli (Finger et al. 2000) which is useful in life threatening situations as well as in a competitive reproductive setting. In addition to the MOE, there is a specialized olfactory organ, the vomeronasal organ (VNO) that is used for pheromone detection (Wyatt 2006 ). Vomeronasal Organ The pr edominant proposed use of the VNO is for reproductive signal reception. The VNO projects to the accessory olfactory bulb which then projects to regions of the amygdale distinct from the targets of the MOE ( Finger et al. 2000 ). There are two known receptor types of the VNO; type 1 (Dulac and Axel 1995) and type 2 (Herrada and Dulac 1997, Matsunami and Buck 1997) (V1R and V2R respectively). Both are seven transmembrane g protein coupled receptors ( Halpern et al. 1995 ). The direct link from the accessory olfa ctory bulb to the amygdala and subsequently to subcortical pathways ( Figure 1 2) allows for a faster and less consciously controlled reaction than traditional olfaction. In the past this complete separation of the projections of the main and olfactory bul b s indicate d that the VNO transmits different information than the MOE in most cases pheromonal social cues ( Finger et al 2000 ). However it has become

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21 evident that there is much overlap between the signals and resultant effects mediated by the VNO and MOE (Brennan and Zufall 2006 Wang et al 2007 ) Figure 1 2. The transduction pathways of the VNO and MOE within a hamster. Text and Image Dr. Michael Meredit h and Neuroscience Program FSU. The VNO is thought to have evolved from an extra fold that a ppears in the olfactory bulb of archaic fish species. Over evolutionary time this fold evolved into a separate structure with divergent pathways from that of traditional olfaction and is most developed in terrestrial vertebrates. However, in many reptiles that evolved from a terrestrial to an aquatic existence it is apparent that the VNO is not present or is vestigial. This indicates that species which transitioned from a terrestrial to an aquatic environment have not developed a VNO for use in aquatic sy stems (Bertmar 1981, Eisthen 1992). In addition, birds and primates, including humans, do not have a functional VNO (Meisami and Bhatnagar 1998). Taste Taste reception is predominantly associated with food preference and is a combination of several sense s including texture, olfaction and the detection of spice or

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22 chemesthesis. Th is combination of senses and the ability to respond to a wide variety of signals ranging from large lipid molecules to small simple chemical compounds such as salt makes taste uni que compared to other senses. Taste buds are the primary end organ for taste in vertebrates and are found predominantly on the tongue in various papillae as well as in other areas of the mouth including the soft palate, upper esophagus and epiglottis. Ther e are five known types of taste. Salty and sour are detected through ion channels S weet, bitter and umami are detected through G protein coupled receptors. There are two genetically distinct taste receptors: type 1 (Li X et al. 2002) and type 2 (Adler et al. 2000, Matsunami et al. 2000) (T1R and T2R respectively). In general there are more T2R genes, also known as bitter taste receptors than T1R, sweet and umami or protein receptors. There is a correlation between what an animal eats and the number of T1R and T2R genes. For example, herbivores such as cows have more bitter receptors than carnivores such as dogs most likely to protect them from toxins in vegetation (Nei et al. 2008). In mammals, taste buds are composed of at least four types of taste ce lls that each appears to be responsible for various forms of signal reception. This varies according to the organism. These cells can be distinguished by their morphological characteristics including shape and cytoplasm color. Type I cells appear to be gli al like: they support other cells and provide ion gradient control for the sour and salty senses T ype II cells contain receptors for sweet, bitter and umami transductions. Type III cells are an intermediary between receptors and nerve fibers and respond b roadly to sweet, salty, sour, bitter and umami signals that are picked up by type I and II cells The

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23 function of type IV cells is not known but they are located a distance from the pore of the taste bud (Suzuki 2007 Chaudhari and Roper 2010 ). Taste bud s synapse on axons of the facial (CN VII), glossopharyngeal (CN IX) or vagus (CN X) nerve for transmission of signals to the central nervous system. These nerves transmit information from rostral (CN VII) intermediate (CN IX), and caudal (CN X) portions o f the oral cavity respectively. Cranial nerves VII, IX and X synapse on the nucleus of the solitary tract (NST). Axons from the rostral, gustatory division of the NST terminate in the parabrachial nuclei which then project to limbic structures including the lateral hypothalamus, central nucleus of the amygdale, and the bed nucleus of the stria terminalis ( Figure 1 3) Portions of the limbic system are implicated in taste perception ( Finger et al. 2000 ) but there are currently no comparative data on change s in limbic and taste CNS structures as has been reported in relation to the olfactory system In addition to taste buds there are solitary chemosensory cells (SCC) that are similar to taste cells but are not located within taste buds. In aquatic sp ecies t hese cells have been located throughout th e exterior body or in olfactory epithelium. In both aquatic and terrestrial invertebrates and vertebrates these cells are located in the oral cavity or within respiratory epithelium, and along the trachea (F inger et al. 2003; organs, and depending on location are responsible for a multitude of sensory reflexes (Hofer et al. 1996; Hofer and Drenckhahn 1998). Like olfactory receptors, require an aqueous environment and so contain either a thick mucus, electron dense substance or are located in a fluid medium such as an aquatic environment. synapse onto distal processes of remote ganglion cells

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24 and a re innervated by any cutaneous nerve including spinal and trigeminal ( Whitear 1992 ). In an aquatic environment, t hese cells are typically involved in predator avoidance and habitat recognition responses, although they have also been implicated in food asse ssment (Kotrschal et al. 1997). Within terrestrial mammals they are used for airway protection and reaction to other abnormal changes in chemical composition of the digestive tract (Hofer et al. 1996; Hofer and Drenckhahn 1998; Finger et al. 2003) Figure 1 3. Transduction of signals to and within the brain during taste with food in the oral cavity. Air flows indicated by dashed and dotted lines; dotted lines indicate air carrying odor molecules. ACC, accumbens; AM, amygdala; AVI, anterior ventral insular cortex; DI, dorsal insular cortex; LH, lateral hypothalamus; LOFC, lateral orbitofrontal cortex; MOFC, medial orbitofrontal cortex; NST, nucleus of the solitary tract; OB, olfactory bulb; OC, olfactory cortex; OE, olfactory epithelium; PPC, posterior parie tal cortex; SOM, somatosensory cortex; V, VII, IX, X, cranial nerves; VC, primary visual cortex; VPM, ventral posteromedial thalamic nucleus. Adapted from Shepherd 2006.

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25 In general, terrestrial and aquatic taste systems are very similar. In both systems taste buds are located inside the mouth. However, there are examples from aquatic species including catfish barbels and lip taste buds in which taste buds are located outside the oral cavity The connections from the receptor cells to the central nervous system are also similar. In fish, t hose taste receptors outside the mouth that are used for food localization synapse on the facial nerve whereas those inside the mouth that trigger swallowing are mediated by the vagus nerve (Finger 1997). In summary, t he signals perceived by the olfactory epithelium, VNO and taste buds can result in changes to behavior and physiology. Signals given off by an animal and perceived by another can be used for communication. In higher order terrestrial vertebrates olfaction is the predominant form of reception while in lower order aquatic vertebrates it is taste. It is not known if chemical communication occurs through taste in terrestrial vertebrates (Wyatt 2006). Chemical Communication Chemical communication can be used with in and between species. Functions include reproduction, alarm, aggregation/recruitment, scent marking/territorial behavior, or social organization. to detect its environment including preda tors, prey, or mates has had limited research ( Finger et al. 2000 Wyatt 2006). In recent years it has been found that increased temperatures and pollutants such as oil (Temara et al. 1999) and copper (Sandahl et al. 2007) can have major consequences to th e senses of taste and smell. In general, a chemical used for detection, communication and engagement between individuals of the same species is termed a pheromone. These signals can both attract and deter potential mates depending on the age and condition of the animals involved. Chemicals

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26 used for signaling are usually leaked metabolites and so are energetically cheap to produce and broadcast which is why they are most likely ubiquitous among species to transmit reproductive signals (Bigiani et al. 2005). Pheromone s The term pheromone comes from the G reek words pherein to carry, and hormon to excite. Initially pheromones were considered externally excreted hormones that acted between individuals. However, the chemical compounds that make up pheromones ar e much more complex than a single steroid hormone. The best way to specify the definition of pheromone is to break it into several categories of chemical signals that represent points on a continuum. The first is the traditional definition of a pheromone a s a single chemical compound that elicits a behavioral or physical change in an individual. The second is a pheromone blend that consists of a mixture of structurally related compounds that only cause a change when in the correct ratio and the final catego ry is a mosaic signal in which many compounds are needed in the right proportions to observe an effect (Johnston 1998 ). To aid in signal detection for terrestrial species proteins are frequently necessary as accessory components. They can be used as odora nt/pheromone binding proteins that transport an odor across the sensilar fluid of the olfactory epithelium or odorant degrading proteins which clear odors out of the fluid ( Vogt and Riddiford 1981, Pelosi 1994 ). There are many different situations in whi c h a pheromone can be used, and d epending on the category or type of response elicited the chemical compounds used can vary. For example many sex pheromones are considered to have evolved from leaked hormones that the opposite sex uses as a signal of impe nding reception (Sorenson and Stacey 1999). Also, the opposite sex can take advantage of the signals

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27 that are used in other areas, such as food perception. For example, t he male oriental fruit moth attracts females using plant derived chemicals that exploi response to food (Lofstedt et al. 1989). Pheromones can have immediate effects on receiver s known as a releaser effect s or elicit long er term physiological changes termed primer effects The first mammalian pheromonal signals to be discov ered were those that have an immediate or releaser effect on the behavior of the receiver such as the expression of a volatile chemical used by female elephants to attract males ( Rasmussen 1999 ). Primer effects include the stimulation of sperm production i n fish which acts by stimulating hormone excretion or termite caste determination which also acts through hormones but instead of adults it affects levels of juvenile hormone development (Wyatt 200 6 ). A single pheromone may act as both a releaser and/or primer and so the reactions can be very complex (Sorenson 1996). Pheromones used by one sex to attract the opposite sex for the purposes of reproduction are known as sex pheromones and most likely evolved through means of sexual selection (Wyatt 200 6 ). In sects have specialized neural circuits known in moths as the macroglomerular complex to process sex pheromones. Terrestrial v ertebrates use a combination of the vomeronasal organ (VNO) and main olfactory epithelium (MOE). These are both scent structures that receive chemicals in the environment ( Finger et al. 2000 ). Sex pheromones convey a combination of information to potential mates includin g receptivity, species, sex, genotype, and even health. These signals can be used by species with internal fertilization to allow females to choose the most beneficial males such as in the scorpionfly, Panorpa in which males provide food to

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28 females and br Thornhill 1979). Sex pheromones also are used to coordinate external fertilizers such as observed in the mass spawning of corals that in part may be triggered by steroid hormone release (Twan et al. 2006). In contrast to sex pheromones in which an individual is attracted to signals generated by the opposite sex, aggregation pheromones incite resp onse to a signal from either sex. These attracting pheromones are important in many invertebrate species both ma rine and terrestrial. This could be useful for the recruitment of larva such as in barnacle s Balanus amphitrite which use a species specific settlement inducement protein complex to attract larva to a particular settlement area (Dreanno et al. 2007). Ano ther form of aggregation pheromone is used by the tree killing bark beetle, Pityogenes bidentatus to attract large numbers of the beetles to host trees thereby overcoming the tree s defenses. T o stimulate aggregation, t hese beetles use pheromones contain ing methyl and alcohol groups and host tree component chemicals as well as chemicals given off by symbiotic bacteria in the digestive system of the beetles (Byers 1995). On the other hand species use aggregation pheromones to group together as defense aga inst predation, such as in the California spiny lobster, Panulirus interruptus (Zimmer Faust et al. 1985). Another form of aggregation is used by males of a species to respond to male sexual pheromone signal. The signal is initially intended to attract fem ales but aggregation occurs when other males respond as well (Wyatt 200 6 ). Alarm signals result in a fight or flight response by an individual which can be in response to predators or invasion of a nest. These signals are not as altruistic as the

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29 name implies. The pheromone given off by a prey species serves to both deter predators and cause a change in behavior by conspecifics to increase potential escape by the signaler. Thus the signal may result in another individual falling prey to a predator becau se of sudden movement or lack thereof. These signals can be used for multiple purposes. For example the ant Formica rufa use s a combination of formic acid and saturated hydrocarbons to defend and alarm nest mates to danger, recruit nest mates or repel e nemies (Lofqvist 1976). The sea anemone Anthopleura elegantissima releases an alarm pheromone, anthopleurine, when attacked to cause contraction of the tentacles of surrounding anemones (Howe 1976). Alarm pheromones also serve to make prey unpalatable an d many species respond to compounds given off in the blood which indicates an attacked conspecific (Wyatt 200 6 ). Scent marking uses specialized glandula r secretions to mark a territory Territoriality is especially effective in social insects because the colony can be in several places at once and therefore can defend a relatively large area. Ants will also defend trails that lead to valuable resources (Holldobler and Wilson 1990). Although some social insects do use pheromones to mark or defend territory this is predominantly a feature of mammals and ve rtebrates in general (Wyatt 2006 ). It may also be that the territorial pheromone use of insects and aquatic invertebrates has not been fully examined yet since the scent marking of mammals is such an obvious behavior. The best example of pheromone use for social organization comes from the honeybee, Apis mellifera There are several pheromones used by the honey bee to maintain the hive. The most important of these is a signal excreted by the queen known as q ueen mandibular pheromone (QMP) that is made up of five principal chemical

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30 components (Winston and Slessor 1992). The pheromone is relatively nonvolatile and is incorporated into the colony by messenger bees that pass the message on through antennal contac t with other bees in the hive. It indicates that a bee is a member of that nest and inhibits reproduction by other bees. The pheromone is also short lived and is continually produced by the queen. If the queen dies, then the pheromone is no longer availabl e and production of a new queen will begin. This pheromone is considered an honest signal that allows a hive to function efficiently with only one queen. QMP is used by many social insects including other bees Apis sp. and fire ants Solenopsis invicta ( Var g o and Laurel 1994 ). In all of the pheromones described above it is very important to have a differentiation between species to prevent inappropriate mating, and in individuals to prevent attack on a member of the nest or family. For this reason animals have developed the ability to discriminate between very small variation s in chemical structure T he pheromone blends or mosaics used can be extremely complex ( Finger et al. 2000 Wyatt 2006 ). The study of the development and evolution of these chemicals a nd receptors provides fascinating insight into phylogenic relationships as well as divergent and convergent evolution. The expression of chemical signals can occur through normal processes such as urination and defecation or can involve dedicated structure s such as anal glands. The use of chemical signals is ubiquitous among aquatic and terrestrial animals. The type of signals used to broadcast information differs because of the variation in chemical behavior between terrestrial and aquatic environments. Pheromones in terrestrial systems are generally small, volatile compounds such as acetates or acids. Those used

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31 in aquatic systems are also small but are not volatile and are easily transported in water, for example small peptides or steroid hormones (Wyatt 200 6 ). These principles nonvolatile because it is passed through contact and not the air. Although generalizations can be made about terrestrial versus aquatic and the purpose of certain chemical signa ls, the exact composition of those signals is usually very complex and there are still factors of pheromone mixtures and mosaics that are not completely understood today. Mammalian Reproductive Chemical Communication Mammals have a wide variety of chemica l signals to broadcast their reproductive status to the same and opposite sex. One of the most interesting component s of pheromone mediated mate choice in mammals is the ability to detect single locus changes in major histocompatibility complex (MHC) genes MHC genes, known as human leucocyte antigen (HLA) in humans are a component of the immune system. Differing smells in human body odor and urine caused by changes in MHC can distinguish parents, siblings, half siblings and 50% of cousins from non related mates (Wyatt 200 6 ). The more variation between MHC/HLA in potential mates, the more likely a mating is to occur. In humans the stronger the variation the more attraction there is between potential mates (Wedekind et al. 1995). There are several specific effects of male and female excretions that influence the reproduction of female and male mammals. These effect s which include the Bruce, Whitten, Vandenbergh, Lee Boot, and Coolidge effects have been demonstrated both in laboratory animals such as mice an d hamsters and in natural settings. The Bruce effect is a pregnancy block discovered in mice that results from females smelling urine

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32 released by males. The scent of almost any male that did not fertilize the eggs of the female will result in a block of i mplantation of the fertilized eggs (Bruce 1960). The Lee Boot effect is a female urinary pheromone mediated signal that inhibits estrus in adult female mice and delays puberty in juvenile female mice. This effect occurs in high density situations where mal es are limited (Vandenbergh 1999).The Whitten effect results in the induction of estrus in adult females by male pheromones while the Vandenbergh effect accelerates puberty in young females through the same male pheromone signals (Ma et al. 1999, Novotny e t al. 1999). Finally the Coolidge effect increase potential variability in genes (Johnston and Rasmussen 1984). Also, in populations that have a dominant and subordinate female the dominant female will 6 ). In the common marmoset, Callithrix jacchus this occurs through a combination of olfactory, visual and behavioral cues (Barrett et al. 1990). As described earlier, t he VNO is a specialized structure located in the rostral area of the head directly above the palate (Bertmar 1981). Various behaviors observed in mammals are used to deliver scents to the VNO ; the most common is termed flehme n Flehm e n is a physical movement of the lips and tongue that allows for easier access of chemicals to the VNO (Ladewig and Hart 1980) It has been observed in many terrestrial mammals including goat (Ladewig and Hart 1980), horse (Stahlbaum and Houpt 1989), domestic cat (Bland 1979) and dogs (Dawley 1998). The opossum uses a behavior known as snuggling in which the snout is passed over an odor source

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33 several times (Poran et al. 1993). Each mammal has its own method for directing sc ents to the VNO including flehme n, snuggling or some other behavioral technique. Until recently the VNO was considered the processing center of pheromones while the MOE was for receiving traditional scents. However, it has recently been discovered that pheromones can be perceived by the MO E and therefore species without a VNO can respond to pheromonal cues (Kelliher 2007, Zufall and Leinders Zufall 2007). It may be that in species with a VNO this structure is necessary for the initial sensory perception but once a response to signal has be en established the MOE is adequate as has been observed in the pig (Dorries et al. 1997). There are a multitude of behaviors that result from pheromonal cues in mammals. These are essential to the individual broadcasting or responding to the cue in order to perpetuate their genetic material as efficiently as possible. These cues are thought to have evolved as a result of sexual selection over evolutionary time ( Sorenson 1996, Rasmussen 1999 ) They are not unique to mammals but the variety and complexity of these signals in mammals make the specific chemical combinations and resulting behaviors difficult to study. More research is needed on how those effects observed in laboratory settings correlates with natural conditions and on the specific composition of each chemical signal. Very few mammal species have been studied in depth for their use of sexual pheromonal cues. Most of these studies have been based in the l aboratory with such species a s the mouse, rat or hamster. By comparison, the elephant is the predominant example of pheromonal cue use in a wild species Paenungulata One of the most interesting phylogenetic relationships is contained in the clade Paenu n gulata which includes three extant orders of mammals: Proboscidea

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34 (elephants), Sirenia (mana tees/dugongs), and Hyracoidea (hyrax) (Simpson 1945). To understand these relationships development and current common features. The genetic relatedness of these orders has been exhibited in hemoglobin sequences (Kleinschmidt et al. 1986) and chromosome painting (Pardini et al. 2007) while their common diet, vibrissae (Reep and Bonde 2006), lingual structure (Yoshimura et al. 2008) and many other physical characteristics are observable. Despite the l arge differentiation between an aquatic and terrestrial lifestyle the manatee and elephant have a surprising number of characteristics in common. The most obvious is the herbivorous diet, which indicates similar taste structures to allow for detection of n utrients or toxins in their food. They also have a similar mother calf pair bond in which the mother feeds the calf from axial mammary glands until it is about two years of age (Marmontel 1995, Emanuelson 2006). However, unlike manatees, elephants have a tightly knit social structure in which the calf is cared for by other adult females in the herd known as allomothering and has contact with its siblings and other calves. Elephants also remain in the herd well beyond weaning; for their entire lives if fema le and up to 20 years if male (Lee 1987). Manatee calves remain with their mother for 2 3 years and although they follow similar migratory routes do not overtly socialize with the mother after weaning (Marmontel 1995). Although social herds are common in elephants and not in manatees there is a similarity in the dispersed male female social groups of elephants and the complete lack of male female sociality except during reproduction in manatees. This absence of continuous sociality results in reduced male male competition and therefore males

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35 project their status and condition to females using other methods. Elephants tend to move in a home range that changes according to rainfall, vegetation or reproductive receptivity with a variation of 34 km 2 6400 km 2 depending on the herd (Schulte 2006). The manatee predominantly migrates to warm water sites in the winter and vegetation dense areas in the summer with about 12% remaining resident in a site year round The manatee is mostly reacting to changes in temper ature for its migration but vegetation and perhaps, mate localization also affect migration (Deutsch et al. 2003). In elephants the temporal glands on the side of the male musth signal which in combination with behavior indicate s condition and position in the hierarchy (Schulte and Rasmussen 1999). A component of this broadcast signal is the volatile chemical frontalin. As an elephant ages it develops differing ratios of the enantiomers or mirror image of the chemical structure for frontalin which initiates increased interest from dominant females. Therefore, an older and higher condition male will have more mating access to dominant females than the younger males (Greenwood et al 2005). These temporal glands and the musth produ ced in them are unique to the elephant, although many other species use glandular excretions to express their reproductive status. Manatee males may use anal or other glands to express their condition and female s may broadcast their reproductive state to a ttract males. To detect signals expressed in urine or other excretions, e lephant s have a distinct flehme n response in which the elephant touches a sample with the trunk and then places the trunk tip in their mouth exposing the opening of the VNO to the sample of concern (Rasmussen et al. 1982). This adaptation allows the elephant to direct

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36 scents of interest directly to their VNO and this results in more control over the scents on which an individual may focus. The manatee has also been known to exhibit f lehmen like movement of their muzzle but absence of a VNO indicates that this is likely for another purpose. Elephants use infrasonic communication and have excellent hearing and vision (Langbauer 2000) while manatees have limited near sighted vision an d adequate their predominate form of sensory perception (Reep et al. 2002, Reep and Bonde 2006). Their sensitivity to touch may en able infrasonic sound detection like the elephant but there is no direct evidence to support this The lack of social interaction between female manatees precludes them from female bonding pheromones (Rasmussen 1999) or the extensive vocaliza tions that keep the herd organized and functioning (Langbauer 2000). Also, the Asian elephant ( Elephas maximus ) has matriarch le d herds that utilize information passed from generation to generation on food and water location (Rasmussen 1999). Although the female manatee remains with her calf for 2 3 years and does pass on information about food and water, the breakup of this bond does not allow for the continued learning that is found in elephants. Therefore the need to taste salt water gradients and diffe rences in food sources is greater in the manatees than elephants because of the decreased time to acquire learned sources from the mother This would indicate a higher preference of taste for manatees while elephants have a much higher emphasis on smell.

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37 T he elephant has a highly developed VNO and olfactory system (Johnson and Rasmussen 2002, Gobbel et al. 2004) unlike the manatee which does not have a VNO and has a reduced olfactory system ( Lowell and Flanigan 1980 Mackay Sim 1985, Reep et al. 2007). Duri ng reproductive periods male elephants use their VNO and MOE to detect chemical signals given off by the females in urine that indicate receptivity. These signals can include information indicating species, sex, individuality, signals to offspring and othe r females, and a quantitative amount of pheromone signal directed at males (Rasmussen 1999). The use of smell for these purposes by the manatee is 1984) as ha s the possibility of an additional chemical sense such as taste or external receptors (Vosseler 1924, Hartman 1979). Very little chemical communication research has been completed on members of the Paenungulata taxon other than the elephant The hyrax is known to have a si milar set of vibrissae to the manatee that could be used for both detecting their environment or communication (Sale 1970). The hyrax also has a vomeronasal organ (Stobel et al. 2010) and a gland on the dorsocaudal area of their back that has been linked t o reproduction and attention from the young (Olds and Shoshani 1982). Unlike the manatee the hyrax live in family groups and unlike the elephant these are usually male lead, similar to rodents. In general, the importance of chemical communication to eleph ant reproduction and social hierarchy is the example by which future studies of Sirenian and Hyracoidean research should follow. Aquatic Mammals Chemical senses were the first sense to appear in early evolution during the period of time when all species o ccupied an aquatic environment. Taste and smell

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38 developed within those individuals and are evident in varying forms within archaic and derived aquatic species today. Variation in sensory use can occur for a variety of reasons including status in the food h ierarchy, temperature, humidity, rainfall, or the medium in which a species lives. The largest effect on the function of the chemical senses is water. An aqueous as compared to terrestrial environment affects how chemicals are distributed and available for reception. Therefore the greatest differences in taste and smell organ structure and function can be observed between aquatic and terrestrial species. This is most evident in the decreased or absent sense of smell observed in marine mammals (Freitag et a l. 1998). Olfaction The pinniped olfactory system is less developed than terrestrial carnivores (Oelschlager and Oelschlager 2002; Reep et al. 2007, Montie et al. 2009) and phocids and walrus Odobenus rosmarus have a reduced olfactory bulb compared to otariids (Harrison and Kooyman 1968). However, this is based on preliminary examination of a limited number of pinniped brains and more detailed descriptions are needed because these results tend to contradict beha vioral observations (Kowalewsky 2006). For pinnipeds, olfaction is most likely important in mother pup interactions (Phillips 2003) and perhaps foraging. Their ability to follow a direct tract to foraging ground s, even though the position of those location s change within a large area led researchers to believe that chemoreception was being used. A study using two harbor seals, Phoca vitulina indicated that they can detect picomolar concentrations of dimethyl sulphide, a chemical expressed in highly produc tive foraging areas of the ocean (Kowalewsky 2006). Also, territorial marking is thought to occur in the ringed seal, Phoca hispida using facial glands. The combination of alcohols and lipids within

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39 the samples indicate that they can be deposited on surfa ces and detected as volatiles through olfaction (Ryg et al. 1992). The trend towards a reduced olfactory bulb in p hocids and walrus may be linked to the amount of time they spend with their young. For example, the hooded seal, Cystophora cristata a p ho cid, weans its pup in four days and does not leave them during this time (Bowen et al. 1985) ; w hereas the South American fur seal, Arctocephalus australis nurses for up to twelve months and will leave its pup for days at a time while foraging. B ehavioral observations indicate that fur seals smell their pup upon return and before nursing to identify them (Phillips 2003). Walrus nurse at sea and are the only pinniped known to forage with their young (Fedak et al. 2002) which would preclude the necessity of reunions following foraging Therefore olfaction is likely less important in phocid and walrus mother pup interaction s but their general use of ol faction should not be dismissed. N o research has focused on pinniped u se of chemoreception in the aquatic environment for the purposes of territoriality or reproduction although anecdotal evidence indicates this may be the case (Reeves et al. 2002). Anatomic evidence indicates that o dontocetes do not have olfactory capabilit ies. The few o dontocete species examined do not have an accessory and olfactory bulb, olfactory tract or anterior olfactory nucleus as adults (Oelschlager and Buhl 1985, Marino 200 7 Oelschlager et al. 2008a). The regression of the odontocete olfactory sys tem has been demonstrated through histologic examination of 13 harbor porpoise, Phocoena phocoena, embryos and fetuses. At the 28.6 mm stage the olfactory fibers were fewer and the olfactory foramina in the cribiform plate smaller than at the 24 mm stage. As the fetus develops the distinction between olfactory fibers and the terminal

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40 nerve becomes more difficult to identify and at 167 mm total length the cells are in rapid regression (Oelschlager and Buhl 1985). Therefore, it appears that the bulb may have been present in early evolutionary lineages of the delphinids and is lost in present day species during embryonic development (Oelschlager and Buhl 1985). In addition to the regression of the olfactory neuroanatomy, one histological study characterizing t he nasal complex of fifteen harbor porpoise, Phocoena phocoena found no evidence of olfactory epithelium (Prahl et al. 2009). There are no published studies on the behavioral ability of the odontocete s to react to olfactory cues. In addition to the anato mic evidence, the investigation of the odontocete genome for olfactory receptor genes corroborates gradual evolutionary loss of olfaction in o dontocetes. Using eight species of toothed whales, a multigene tree of 115 olfactory receptor sequences from a di verse array of class II olfactory receptor paralogues was defined (McGowen et al. 2008). Several independent pseudogenization events support the theory of the loss of an olfactory sense over evolutionary time. This genomic and anatomic evidence in addition to the lack of behavioral accounts of o dontocetes using olfaction indicates that between the Miocene and present day these species lost the ability to smell. This is invariably due to the lifestyle and environment of these animals in addition to the ener getic cost required to maintain an olfactory system. Within the baleen whales, Mysticetes, Oelschlager (1992) examined embryos and early fetuses from three different species ; blue, Balaenoptera musculus fin, Balaenoptera physalus and humpback, Megaptera novaeangliae whale. Unlike Odontocetes the olfactory bulb, tract, and epithelium of the Mysticetes did not regress and was well formed and evident in a 105mm crown rump length ( CRL ) fin whale fetus

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41 (Oelschlager 1992). In addition to the embryologic evidence, of adult brains from 11 Bowhead whales, Balaena mysticetus (Duffield et al. 1992) and a single Humpback (Breathnach 1955) contained olfactory peduncl es, nerve fibers and small holes in the cribiform plate of the ethmoid bone which would allow t he olfactory nerve to pass into the brain. In a recent study, the Bowhead whale olfactory bulb was located and measured 0.13% of the brain weight and 51% of the olfactory receptor genes were intact (Thewissen et al 2011). In addition to the embryologic ev idence, adult brains of 11 Bowhead whales, Balaena mysticetus (Duffield et al. 1992) and a single h umpback (Breathnach 1955) demonstrate the presence of olfactory peduncles and nerve fibers but no olfactory bulb. The inability to find the bulb is presumed to be due to the size and condition of the available brains ; its presence is conjectured because of small holes in the cribiform plate of the ethmoid bone which would allow the olfactory nerve to pass into the brain (Duffield et al. 1992, Breathnach 1955) In a recent study the olfactory bulb was located and measured 0.13% of the brain weight. In addition, 51% of the olfactory receptor genes were intact (Thewissen et al 2011) Anecdotal behavioral evidence and a highly reduced but intact olfactory system i ndicate that Mysticetes retain some sense of airborne smell (Oelschlager 1989). Baleen whales spend a majority of their time at or near the surface to feed, unlike their toothed counterparts that dive deep to locate prey (Berta et al. 2006). Therefore the use of airborne scents to locate groups of zooplankton or other prey as well as perhaps locate signals given off by predators or mates would correspond with the anatomic evidence of the Mysticetes. It would be interesting to see if there is a variation in olfactory size

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42 between those Mysticetes that skim off of the surface such as the bowhead and those that feed on invertebrates from bottom sediment like the gray whale (Berta et al. 2006). Berta et al. (2006) state that S irenians have a larger olfactory bu lb than cetaceans and pinnipeds ; h owever Reep et al. (2007) demonstrated that manatees have a smaller olfactory system than pinnipeds. This study did not include comparison s to dugongs or cetaceans and it would be interesting to complete the comparison us ing representatives of these taxa Taking into account the habitat of the respective marine mammals that s irenians would be expected to have a larger olfactory bulb than Mysticetes but smaller than pinnipeds. This comparison correlate s with the aquatic lifestyle of Sirenians and with what has been observed in other marine mammals. In this regard a portion of the brain is still dedicated to the processing of scent (Reep et al. 2007) and since the manatee has limited opportunity to take in airborne scent s, e.g. only during a breath, then this center may be adequate for the manatee to process important scent information during feeding, copulating, and simultaneous breathing with other animals. Vomeronasal Organ There are limited anatomic studies examinin g the olfactory capabilities of p innipeds. Although i t is widely reported that they possess a VNO (Kowalesky et al 200 6 Mackay Sim et al. 1985, Meisami and Bhatnagar 1998) no references have an examination of the pinniped VNO or its development. Most comm only referenced information on the VNO in pinnipeds include a note on the pinniped olfactory bulb but do no t mention a vomeronasal organ or accessory olfactory bulb ( Harrison and Kooyman 1 968 Oelschlager and Oelschlager 2002 ) In addition, an examination of the brain of a live Californian sea lion using magnetic resonance imaging gave no indication of a

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43 vomeronasal organ or accessory olfactory bulb, although olfactory structures were documented (Montie et al. 2009). During an examination of lateral olfact ory tract fibers in relation to the accessory olfactory bulb an accessory olfactory structure was observed in the Alaskan fur sea l, Callorhinus ursinus Eumetropias jubata and California sea lion Zalophus californicus No accessory o lfactory tract was found in the Harbor seal, Phoca vitulina (Switzer et al. 1980). It appears that pinnipeds do have an accessory olfactory bulb and therefore a VNO but more research is needed to compare the development, size and functionality of that orga n to those in other well studied mammals. Odontocetes do not have a VNO. There is no evidence of development in embryos or fetuses in harbor porpoise (Oelschlager and Buhl 1985) nor is there any evidence in adult harbour porpoise (Marino et al. 2001). Myst icetes do not have a VNO or evidence of a vomeronasal nerve during development or as adults (Oelschlager 1989). In Sirenia, the Florida manatee does not possess a VNO (Mackay Sim et al. 1985 ) and there is no literature examining the dugong However, both m ysticetes (Thewissen et al. 2011) and s irenians ( Mackay Sim et al. 1985) have a small olfactory bulb I n sheep and pigs, t he female is still attracted to the male and still has a luteinizing hormone surge even if the VNO has been severed (Wyatt 2006). Th is indicate s that marine mammals that have olfactory capabilities may use these to detect and communicate with potential mates even though their VNO is absent. Taste Examination of a California sea lion, Zalophus californianus indicates that p innipeds ha ve a sensitivity to sour, bitter, and salty but not sweet (Friedl et al. 1990). Umami was not tested although they are most likely sensitive because of their diet.

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44 p innipeds are able to detect saltwater gradients within a 4% variance at levels of 30 parts per thousand as determined through a go/no go paradigm of two harbor seals (Sticken and Dehnhardt 2000). According to Yoshimura et al. ( 2002), the California sea lion has taste buds on vallate and fungiform papillae. Although behavioral evidence indicates that pinnipeds do have a sense of taste, more definitive evidence of taste bud location, structure, density and size is needed. In a single sample from a young, 1.5 year old, male walrus it was observed that there are very few (140 or less) but large (75 X125 m) taste buds on the walrus tongue (Kastelein et al. between the harbor seal with the greatest amount and the California sea lion and grey seal with the least (Sonntag 1923). The w alrus use s a suction feeding method and so it is likely that mechanoreception is more important than gustation. However, captive walrus es do exhibit selectivity in food choice and so may be using taste under certain circumstances (Kastelein et al. 1997) Bottlen ose dolphins have taste buds in pits at the base of their tongue ; these pits d evelop from circumvallate papillae that are present in neonates (Kuznetzov 1990). Also, t he y have a highly developed gustatory nucleus of the thalamus (Kruger 1959).Go/no go paradigms using a single adult captive male have indicated that bottlenose dolphins can respond to the four common tastants; salty, sour, sweet and bitter at an order of magnitude below human capabilities. Another study using a similar paradigm demonstrate d that a single 3 5 year old male dolphin had a threshold of .0173M for a sour tastant and 1.36X10 5 M for bitter, comparable to human thresholds (Nachtigal and Hall 1984). Umami has not been tested.

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45 Several Russian researchers have hypothesized that becau se of the limited number and specialized structure of dolphin taste receptors they may use taste for chemical communication (Friedl et al. 1990). However, no form of chemical communication has been tested for using dolphins. Other Odontocete species have been examined for taste buds with mixed results. Harbour porpoises ( Phocoena phocoena ), Amazon River dolphins ( Inia geoffrensis ), and beluga whales ( Delphinapterus leucas ) were discovered to have a novel microvilli structure in the papillae, but no taste b uds were observed (Kuznetzov 1990). No taste buds were found in the Indus river dolphin ( Plantanista gangetica) (Arvy and Pilleri 1970), La Plata dolphin ( Pontoporia blainvillei ), (Yamasaki et al. 1976), the sperm whale ( Physeter macrocephalus ), or the lo ng finned pilot whale ( Globicephala melas) (Pfeiffer et al. 2001). However, the examination of the pilot whale did not include the tongue root, the area where most taste buds are located in cetaceans Taste buds have been located in a newborn male Stejneg Mesoplodon stejnegeri (Shindo et al. 2008) and striped dolphins, Stenella c oeruleoalba, of varying sex and age (Komatsu and Yamasaki 1980). In Baiji river dolphin s Lipotes vexillifer taste buds were located with fewer found in a young adult female than a calf and none in an older adult female (Li 1983). The conflicting evidence of taste bud presence in Odontocetes could be due to a number of factors. T he small size of the tongue and relatively higher density of taste buds in fetal and n ewborn dolphins enables easier identification of taste buds in young animals than the limited number of taste

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46 buds and larger tongue size of adults. Another possibility could be a regression of taste buds post weaning. These examinations involved small sa mple sizes and limited comparisons between age and sex. There were also many instances of taste bud localizations in one taste ability and use is difficult to determine c urrently but in general o dontocetes have the ability to taste and may be using this in food assessment or mate recognition. A combination of behavioral and anatomic analyses of taste buds on the bottlenose dolphin (Friedl et al. 1990) provide the most rel iable and useful results, although not feasible with most species. There are no formal published reports on the use of taste or presence of taste buds in m ysticetes. It is assumed that taste is used by m ysticetes, perhaps more than o dontocetes, since the use of baleen allows for food to be present in the mouth for a longer period of time, giving the animal the chance to expel any undesirable food. The dugong has taste buds in pits of the lateral margins of the tongue (Yamasaki et al. 1980). They live in a marine environment and so may also use taste for freshwater detection and food palatability but it is not known if their chemoreception is used during reproducti ve behavior The Florida manatee has taste buds in the foliate papillae of the tongue root (Levin et al. 2002). In general, S irenian taste buds are more numerous than those of cetaceans or pinnipeds (Yamasaki et al. 1980). There has been no published examination look ing for taste buds or solitary chemosensory cells in any area besides the tongue in any marine mammal I t would be advantageous to have some kind of sense organ outside the mouth to assess

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47 palatability of food, locate mates, and determine salt water gradie nts e specially for those species living in a marine environment in which salt balance is a concern Of the approximately 125 known marine mammals of the world 22 are listed under the endangered species act as threatened or endangered. Over the past 60 ye ars three marine mammals have gone extinct including the most recent Baiji River Dolphin in China. For each of these species the cause of their extinction or vulnerability is in some way human related including pollution, vessel strikes, and bycatch (Ma rine Mammal Protection Act 2010). Considering the precarious situation that these animals are in and the known influences of environmental contaminants on chemoreception, it is vital to learn as much as possible about their biology in order to decrease any negative effects of pollution and/or climate change. An endangered species that provides the opportunity to explore the use of taste in smell in a fully aquatic mammal is the Florida manatee. The Florida Manatee The endangered Florida manatee, Trichechus manatus latirostris presents a unique opportunity to study the use of chemoreception by aquatic mammals. The animals are available in Florida in the wild, as live animals maintained in captivity which are available for behavioral assays and as postmort em carcasses Furthermore, it is one of many marine mammals suspected of using chemosensation for reproductive 1956). The reproductive behavior of the Florida manatee, Trichec hus manatus latirostris is not well understood due to the difficult nature of studying reproduction in a low visibility, aquatic environment of a sparsely populated endangered species. In

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48 addition, the manatee has limited social interactions which includ e mother/calf bonds and mating herds in which many males follow a receptive female for up to four weeks (Hartman 1979, Reep and Bonde 2006). It is the formation of these mating herds that is of interest, as it has been observed that males will travel appre ciable distances to track females in estrus. Although the manatee may mate at any time of the year, peak sexual activity occurs during the warm spring and summer months when manatees have dispersed from their warm water refuge sites (Reep and Bonde 2006). Therefore, it is assumed that there is a signal, presented by the female, which alerts males to her receptiveness. Although this signal could be visual or auditory, the manatee has limited vision and is reported to be near sighted, having vision only slig htly better than river dolphins (Supin et al. 2001). In addition, manatee vocalization generally occurs in mother and calf communication although it has been observed in mating herds (Hartman 1979). Their vocalizations consist of short chirps or squeals in the range of 3 24 kHz (Nowacek et al. 2003) and would most likely be lost in the estuarine environment of the manatee over long distances. A more likely source of this signal would be chemical cues given off by the female. There have been a number of documented behavioral observations of the manatee indicat ing that the female does produce chemical signals. Among the earliest came from Dr. Vosseler, a German veterinary practitioner who was responsible for the care of a male and female manatee in captivi ty together. He noted that when the female was in estrus the male would swim at the bottom of the tank moving his nose and lip, both lateral and medial parts so that the inner lip was exposed. In addition the male

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49 would always find the locations along th of mouthing between manatees and hypothe sized about their chemoreceptive abilities. Furthermore, there has been documentation of manatees in Crystal River, FL rubbing each year on the same submerged logs and stones. These actions were observed to occur most frequently around the genitals, eyes, arm pits, and chin. It was hypothesized that females, which rubbed more frequently than males, were leaving a The sum of the behavioral accounts indicate that a female manatee may be able to broadcast her reproductive state util izing secretions and/or excretions. The most likely source of this signal is urine, as this is the broadcast used most frequently by mammals (Wyatt 2006). Other potential sources include anal or vaginal glands (Marmontel 1988). A steroid or peptide derivat ive secreted in the mucus of the vagina could stick to those rubbing posts and tanks on which manatees have been observed rubbing. Anatomic evidence also indicates that the use of taste and/or smell for reproduction is feasible. The Florida manatee has ta ste buds in foliate papillae located on the side of the tongue root (Levin and Pfeiffer 2002 ) and most likely use taste for detecting salt water gradients or seeking specific nutrients from vegetation (Reep and Bonde 2006). Their herbivorous diet increa ses their vulnerability to toxins that can be detected by a strong bitter taste (Nei 2008) which is generally the function of foliate papillae (Collings 1974). Touch is probably their predominant sense for food detection

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50 but taste is likely supplementary. It has long been thought that males use chemoreception to find female manatees in estrus and so it is not known if they use their sense of taste or smell or a combination to do this (Levin and Pfeiffer 2002, Reep and Bonde 2006). In addition, it has been h ypothesized that the manatee possess modified taste or smell receptors on the lips or roof of the mouth due to their behavior of mouthing each other and objects (Vosseler 1924, Hartman 1979). A lthough the VNO does generally play a role in pheromone detect ion, the MOE is an equally important part of the signal detection and capable of pheromone reception without the VNO (Wang et al. 2007). This may have direct implications for the Florida manatee, especially with regard to animal husbandry. In laboratory mi ce the MOE was implicated in long range detection while the VNO was used during close contact estrus detection (Achiraman et al. 2010). The male manatee may detect female presence from long distances using chemical signaling and within close contact using touch. Therefore, t Sim 1985) could be the site of these signal transductions and would explain the continued presence of an olfactory bulb (Mackay Sim 1985). In addition, it has been hypothesized that the manatee may possess modifi ed taste or smell receptors on the lips or roof of the mouth due to their behavior of mouthing each other and objects (Vosseler 1924, Hartman 1979). their social actions w hen in a mating herd indicates that they use chemosensation. This could be through taste and/or smell receptors and it is not clear which pathway is most used. The combination of manatee availability relative to other marine mammals in

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51 addition to the beha vioral evidence warrants an in depth examination of their olfactory and taste capabilities. Through histological examination of suspected sites of chemoreception and signal transmission in addition to behavioral observation of the reaction of captive male manatees to female manatee urine chemoreceptive abilities is possible. This will allow for conservation efforts to target those areas of the environment necessary for the highest fecundity. This investigation provide s chemosensory capabilities especially with respect to reproduction. Concurrently, an appreciation of the use of chemoreception in fully aquatic mammals has b e e n documented for the first time.

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52 CHAPTER 2 ANAL GLANDS OF THE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS: A POTENTIAL SOURCE OF CHEMOSENSORY SIGNAL S Background Anal glands have been identified in a variety of terrestrial and aquatic species (McColl 1967) with detailed anatomic description in a limited number of animals (Montagna and Parks 1948, Eglitis and Eglitis 1961, Greer and Calhoun 1966). Anal glands have been observed in all species examined, however, in humans the glands are proportionally smaller than in other animals and appear to have either limited or no function (Eglitis and Eglitis 1961, McColl 1967). All glands from the tissue surrounding the anus and anal canal are a combination of apocrine and sebaceous secretory units and can be found anywhere along the anal canal or in perianal tissue (Eglitis and Eglitis 1961). Many of the glands have been modified from sweat glands. They can be found singly, with many openings into the canal such as in pigs, Sus scrofa (McColl 1967) or the various secretions can collect in an anal sac and be secreted through a single large opening such as in the cat, Felis domesticus (Greer and Calhoun 1966). This duct can end in a nipple, as observed in the black tailed prairie dog, Cynomys Ludovicianus (Jones and Plakke 1981) or as a single opening commonly observed in dogs, Canis familiaris (Montagna and Parks 1948). Secretion by anal glands appears to occur through a combination of myoepithelial cells surrounding the adenomeres (Atoji et al. 1998) and the position of the glands between internal and external sphincter muscles (Montagna and Parks, Jones and Plakke 1981). Therefore, the secretion can be through both voluntary and involuntary muscle movement.

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53 Anal glands are generally accepted to be used for the expression of reproductive chemical signals but secretions can als o be used to identify species, sex, or produce odors for defense or subduing prey (Eglitis and Eglitis 1961). Anal glands have never been associated with a function other than those related to chemosensation. Animals that use anal glands for reproductive c ommunication typically produce varying amounts and types of secretions in relation to their reproductive cyclicity (Gorman et al. 1978). Previous research on anal glands of terrestrial animals demonstrated their use for territorial marking in canines (Dono van 1969), the beaver, Castor fiber (Rosell and Schulte 2004), and the Eurasian river otter, Lutra lutra (Gorman et al. 1978). Although the beaver and river otter inhabit both terrestrial and aquatic environments they appear to use these secretions only o n land (Rosell and Schulte 2004, Gorman et al. 1978). In fish, anal glands are used by males of the Blenniidae family for reprodu ction. Several species of male b lennids have a pair of anal glands found on either side of the anal fins and behavioral testing has demonstrated female mate choice is based on the presence of these glands and their contents (Barata et al. 200 8 ). The function of mammalian anal glands in an aquatic environment has never been examined. The Florida manatee, Trichechus manatus latirost ris is an endangered fully aquatic herbivorous mammal in the order Sirenia. During a routine necropsy of a young male manatee there appeared to be glands near the anus. This initiated an examination of this region for glands. Anal glands have never bee n described in the manatee or any other fully aquatic mammal although there has been reference to perianal glands with ducts emptying directly into the water of the Black Sea bottlenose dolphin (Kuznetsov 1974) but no further detail was provided. This, in addition to a reference to mucous anal

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54 glands in the common seal, Phoca vitulina (McColl 1967) are the only published accounts of anal glands in cetaceans or pinnipeds. Manatees will be the first fully aquatic mammal to have their anal glands morphologica lly characterized in detail. The goal of this study is to determine the structure of the anal glands and generate functional hypotheses through histochemical analyses a nd comparison to other species. Materials and Methods Samples were collected from freshl y dead manatees that died from boat strikes, Pathobiology Laboratory in St. Petersburg, FL. Eleven animals, six female and five male, ranging from a fetus to a large adu lt were examined (Table 2 1). All animals were in good body condition except for three chronically ill females injured by watercraft that were emaciated: MEC0983, LPZ102765, and LPZ102654. Table 2 1. Samples used for anal gland analysis NBF=Neutral Buff ered Formalin. All information is available from the Marine Mammal Pathobiology Laboratory Manatee Mortality Database1. Adults were measured as a straight length while the fetuses were crown rump length. Animal Number Sex Age Class/Length (cm) Fixation Cause of Death Date of Death SWFTm0826 F Adult/261 10%NBF Chronic Watercraft Injury October 2008 LPZ102654 F Adult/261 10%NBF Chronic Watercraft Injury December 2008 MEC0983 F Adult/275 10%NBF Chronic Watercraft Injury July 2009 MNE0939 F Adult/290 10%NBF Acute Watercraft Injury August 2009 LPZ102765 F Adult/267 10%NBF 2%Glutaraldehyde Unfixed/Frozen Chronic Watercraft Injury August 2010 MSE0910 F Calf/188 10%NBF Cold Stress January 2009 LPZ102639 M Adult/309 10%NBF Chronic Watercraft Injury November 2008 MNW0905 M Juvenile/210 10%NBF Natural March 2009 MSE0943 M Juvenile/205 10%NBF Cold Stress April 2009 M 187 M Fetus/30 10%NBF Aborted Fetus February 1980 F MSW03160 M Fetus/39 10%NBF Acute Watercraft Injury October 2003 1 Manatee mortality database available online at: http://research.myfwc.com/manatees/

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55 Tissue surrounding the anal canal to several centimeters below the anorectal junction was removed. All samples but one wer e fixed in 10% Neutral Buffered Formalin (NBF). From one animal, four one square centimeter sections of anal gland were removed and frozen fresh for lipid staining, while three one cm by one cm by two mm samples were fixed in 2% glutaraldehyde 0.1 M phos phate buffered solution for transmission electron microscopy (TEM) T he rest of the tissue was fixed in 10% NBF. Light Microscopy The anal tissue samples were examined grossly using a dissecting microscope and measurements taken on the size and distributi on of the anal glands. Samples of the glands and ducts were embedded in paraffin and sectioned at 5 30m. The 5m sections were stained with hematoxylin and eosin (H&E) to outline the general anatomy of the tissue, periodic acid Schiff (PAS) to stain glyco gen rich material and identify and the collagen of connective tissue. Sections were mounted on superfrost plus slides and stained using Santa Cruz Smooth Muscle Actin (HUC 1 1) with an Invitrogen Histostain plus kit (AEC) to identify myoepithelial cells. The 20 30m sections were stained with hematoxylin and eosin. Extremely thick sections of approximately 120m were cut on a slab microtome and stained with hematoxylin or tr ichrome to determine the extent of the duct system. Unfixed frozen samples of the anal glands were cut at 6 m using a cryostat and stained with oil red O to identify lipids. Transmission Electron Microscopy Samples fixed in glutaraldehyde were cut into sm all portions (2 mm 3 ) and post fixed in 2 % osmium tetroxide for 2 hours at room temperature. They were dehydrated in grades of alcohol and embedded in an epoxy (Epon Araldite) plastic mixture. Ultrathin

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56 sections between 70 90 nm were sectioned using an ult ramicrotome and mounted on a 100 mesh Formvar coated grid. The sections were stained with uranyl acetate and 7000). Results All of the manatees in this study, regardles s of age or gender had diffuse, large, apocrine glands at the anorectal junction. The anal glands were found within both the internal and external sphincter muscles of the anal canal between 3 and 7 cm cranial to the anal sphincter. They were diffuse and f ound in clusters that cover a large area, reaching 3 5 centimeters in total length and 2 3 centimeters at the widest point. They appear ed to have a tapered shape, forming two diamond patterns between the sphincter muscles cranial to and at the anorectal ju nction They were composed of aggregated glands contained within individual fascia that empt ied into several collecting ducts ( Figure 2 1 ) ranging in size from 3 170 m in diameter. The opening of the duct was not readily visible grossly because of the convoluted folding of the integument ( Figure 2 2 ). Figure 2 1 Adult female (MEC0983), collecting duct (CD) approaching the anorectal junction. Scale bar equals 0.5cm.

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57 Figure 2 2 Ventral view of a male manatee calf with an inset of the current working diagram of anal gland anatomy. Scale bar approximates 1 cm. A ) anal glands, B) external sphincter muscle, C ) internal sphincter muscle, D ) Anal Canal, E ) Anorectal junction, F ) Rectum, G ) Excretory duct, H ) Anal orifice. Both males and females in the study had extensive anal glands. Those glands collected during fall and winter months were smaller with less obvious secretory production such as in LPZ102654 which had small, compact glands without a discernab le duct system. Large, adult animals tend ed to have larger glands and one female, MEC0983, had the largest and most obvious duct system. Like adults, the two male fetuses, estimated mid term gestation, had two sets of glands, one on each side of

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58 the canal that tapered from caudal to cranial orientation. However, unlike adults, one fetal specimen, (F MSW03160), had glands that were completely ventral to the anal canal ( Figure 2 3) while in the other fetus (M 187) they were on either side, cranial and caudal to the canal. Figure 2 3. Anal glands (AG) of male fetus F MSW03160 completely ventral to the canal. From cross sectional cut across the anal canal. Scale bar equals 0.5cm. Light M icroscopy The anal glands of the examined manatee s consist ed of branched tubules ( Figure 2 4) which collect ed in large bilayered ducts that emptied directly into the anal canal ( Figures 2 4 and 2 5). These ducts range d in diameter from 3 170 m, increasing as they approach ed the canal. The glands and ducts are emb edded in both smooth and skeletal muscle. At the secretory portion the epithelium was simple cuboidal ( Figure 2 6). The strong eosinophillic nature of the glands and luminal contents ( Figure 2 6A) indicate d a protein ladened substance. The glands produce d a mucous rich secretion as can be observed in the PAS stain ( Figure 2 6B) while the oil red O stain indicate d that lipids are present in the secreting cells and lumen( Figure 2 5C). Trichrome staining

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59 demonstrate d the encasing of the glands in muscle follo wed by connective tissue as they progress ed toward the lumen of the anal canal ( Figure 2 5D). The myoepithelial cells surrounding the adenomeres can be seen in the anti smooth muscle actin antibody stained tissue ( Figure 2 7). Figure 2 4. Collecting duct (CD) and its contents. H&E of adult female MEC0983. Note the large size and branching of the duct as well as its lining consisting of two cell layers. Scale bar equals 100m. Figure 2 5. Collecting duct (CD) joining anal integument as a n excretory duct. H&E stain of anal glands of MEC0983. Note the Proximity of nerve bundles (NB). Scale bar equals 100m.

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60 Figure 2 6. Various histologic stains of adult manatee anal glands. Scale bars equal 100 m. A. Adult female, MEC0983 H&E. Single la yer cuboidal cells visible. Positive eosin reaction within the luminally stored secretions indicates presence of protein. B. PAS stain of adult female, SWFTm0826. The deep purple reaction within the glands indicates mucus while the small clears spots with in and surrounding the purple stain indicates other material. C. Adult female, LPZ102765. Oil red O stain. The brightly red reaction reveals lipid rich material. D. Adult female, SWFTm0826. Masson trichrome. Red stains muscle and glandular cells, while gre en stains connective tissue. The glands are encased in connective tissue and surrounded by muscle. Figure 2 7. Smooth muscle actin stain of adult female LPZ102765 demonstrating the location of myoepithelial cells. Scale bar equals 100m.

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61 Electron M icroscopy Transmission electron microscopy demonstrate d apical caps separating from the cells into the lumen (Schaumburg Lever and Lever 1975). Cells that appear ed to be actively secreting had fewer organelles apically and change d in morp hology and number of microvilli compared to other cells. Myoepithelial cells, often with indented nuclei, formed an incomplete basal layer associated with the secretory tubules ( Figure 2 8B). Figure 2 8. TEM of manatee anal glands The apocrine secretion is evident because of the Apical Caps (AC) A ) Microvilli (M ), Scale bar 5 m B ) Scale bar 10m, a myoepithelial cell indicated (MYO). Discussion While the sample size was small and did not allow for statistical analysis, there were no apparent difference s between males and females. However, seasonal differences are suggested by the smaller size and less productivity of those samples collected during fall and winter months. The samples collected from adult animals in spring and summer months were the larg est and had the most secretory material within the cells and lumen. This included two animals, MEC0983 and LPZ102765, which were chronic boat strike victims and were emaciated at the time of necropsy. The males

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62 examined were young, or the samples were col lected in late fall (see Table 1) thus in all males examined the glands appeared smaller and had less secretory material than in spring and summer females. Further comparison of specimens of various ages and sex should determine whether gender differences exist and further demonstrate seasonal variation. Manatees do not have distinct breeding seasons but do tend to mate during the warmer spring and summer months throughout the Southern United States (Reep and Bonde 2006). The suggestion of a seasonal change between those animals which died in colder versus warmer months indicates that the animals may be using their glands to communicate with mates. This is similar to the behavior observed in the Eurasian river otter (Gorman et al 1978). However, the otter d eposits its secretions on land, while the manatee would be depositing into an aqueous environment. The large size, productivity, and change with season of these anal glands indicate that they are in use and may play important roles for the manatee. The la rge number of glands and storage ducts observed in close proximity to the epidermis suggests that substantial quantities of secretory material can be released at any given time. The location of the glands between muscle layers allows for secretion of conte nts during defecation. It may be that manatees deposit the secretions into the feces and the buoyant nature of the lipids a llow for transport in the water; w hereas the mucous would allow secretions to remain on submerged objects for longer periods of time. It is not known if the secretions can be actively expressed without defecation. The fact that the portion of the sphincter with which this gland is associated contains skeletal muscle suggests that it could.

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63 Anal fistulas and cancerous tumors associated with anal glands are a problem in cats (Greer and Calhoun 1966) and dogs (Williams et al. 2003) and have been documented in humans (McColl 1967). Of all glands examined in these manatees, none showed evidence of impaction or infection. The relatively smal l and well protected emptying ducts indicate that infections typically observed in other mammals are of limited concern to the manatee. However, attention should be paid to anal gland health during post mortem evaluation of manatees to determine prevalence of these conditions. The size of the fetuses examined here indicate that both were gestationally mid term animals, which in manatees would be approximately 6 months of prenatal development (Rathbun et al. 1995). In humans the development of anal glands be gins in the early second trimester (fourth month) (Eglitis and Eglitis 1961). Therefore, the development of these glands appears to follow similar embryologic development as observed in humans. The manatee fetal glands appear to develop at or near the vent ral surface and may move dorsally to surround the anal canal. However, more specimens are required to corroborate this observation and demonstrate the origination of gland development. The separation of apical caps into the lumen of secretory units demonst rates that these glands are apocrine ( Figure 2 8A). Unlike the anal glands of all previously examined animals such as the dog (Montagna and Parks 1948), cat (Greer and Calhoun 1966), and 20 various species examined by McColl (1967), the manatee has only ap ocrine and no sebaceous glands. This indicates that the glands are used primarily for signal transmission (Eglitis and Eglitis 1961). In addition, there is no

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64 reference to anal glands for which the presumed purpose is not the expression of chemical signals Assuming that these structures produce one or more signals, the manatee should possess the ability to perceive these signals, whether by olfaction or taste. The manatee does possess taste buds (Levin and Pfeiffer 2002) and olfactory epithelium (Mackay Si m et al. 1985) ; however, the extent to which these systems are used is unknown. Behavioral and comprehensive anatomic examination of these systems is needed to determine how the manatee perceives chemical signals. We hypothesize that the manatee uses its a nal glands for chemical communication. As the manatee is not a territorial animal, marking territory is not likely a use for the secretions. Also, the manatee is an herbivore that has no natural predators and therefore would not use the secretion as defens e or to subdue prey. Finally, it has long been hypothesized that these aquatic mammals use chemical signaling for communication and specifically in the broadcast of reproductive signals (Rathbun and cludes documentation of manatees in Crystal River, FL rubbing genitals/anus, eyes, arm pits, and chin on the same submerged logs and stones each year. It has been proposed that females, which rubbed more frequently than males, were leaving a chemical signa l of their reproductive manatee observations include a male In the captive setting, a German veterinary practitioner respons ible for the care of a male and female manatee inhabiting the same tank, noted that when the female was in estrus the male would swim at the bottom of the tank and always find the locations er 1924). The

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65 rubbing of the genital/anal area, which in females are centimeters apart, suggests that a signal is contained in the secretions/excretions of these areas that can be perceived by males. The presence of mucous in anal gland and vaginal secret ions (Marmontel 1988) may increase retention time for these signals on rocks, logs or other surfaces. Although staining can give an indication of whether a secretion contains mucous, lipids, proteins s within that secretion. To determine the purpose of the signals produced and released by these glands, research is needed to determine the exact chemical composition of the gland secretion. Since collection through the ducts from a live or freshly dead an imal is not practical due to the internal design of the duct system a novel chemical analysis method known as matrix assisted laser desorption/ionization time of flight (MALDI TOF) will be used to analyze glands from animals of various sexes and ages to de termine which chemicals are most prevalent in the most productive glands. Through a better a better indication of what type of signal is used to express reproductive sta tus, health, or other parameters to conspecifics.

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66 CHAPTER 3 TASTE BUDS IN THE ORAL CAVITY OF THE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS WITH WITHIN THE LINGUAL ROOT TASTE BUDS Background The sense of taste is mediated which are located predominantly in the tongue but can be found throughout the oral cavity and ep iglottis. Within mammals, t aste buds are generally located in papillae. There are four types of papillae found on the to ngue: fungiform, filiform, foliate, and circumvallate or vallate Except for filiform papillae, which aid in mastication (Iwasaki 2002) taste buds can be found within all or none of these papillae depending on species (Hosley and Oakley 1987) In general fungiform papillae are mushroom shaped and found towards the apex and sides of the rostral portion of the shaped cones lacking taste buds and located throughout the do rsal surface of the tongue Fol iate papillae are p arallel grooves containing large concentrations of taste buds and generally located at the lateral base of the tongue. Circumvallate (vallate) papillae are dome shaped and if present are found on the dorsal surface of the tongue forming a row ( Iwasaki 2002 ) Depending on the location of the taste buds within the oral cavity they synapse on one of three cranial nerves : the facial (CN VII), glossopharyngeal (CN IX) or vagus (CN X) (Finger et al. 2000). In terrestrial animals gustation is a combination of senses including taste, smell, touch, temperature and chemesthesis or spice. In aquatic species, the contribution of volatile odorants to the sense of taste is minimized or absent and in fish the lateral olfactory system cont ributes aqueous chemicals to food detection (Kotrschal 2000). However, in marine mammals, which have no or reduced olfactory

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67 bulbs (Lowell and Flanigan 1980) the sense of taste is likely to have an increased importance to food localization, reproduction a nd communication. The exact transduction mechanisms of taste are not well understood There are four cell types that mediate taste within the taste bud. Type I cells are glial like and support other cells. Cell type II are receptors for sweet, bitter and umami transductions. Type III cells are an intermediary between receptors and nerve fibers, responding broadly to salty and sour ion gradients. The function of type IV cells is not known but they are located near the base of the taste bud and are morpholog ically Merkel like (Finger 2005, Suzuki 2007 ). The main neurotransmitter from taste cells to the gustatory nerves is ATP (Finger et al. 2005) ; h owever, other regulators are thought to play a role in taste transduction including estrogen (Toyoshima et al. 2 007). Within the oral cavity estrogen mucous and salivary gland function (Valimaa et al. 2004) or reproduction (Cheek et al. 1998). Within terrestrial mammals taste buds have b een documented consistently and quantitative measurements taken in a few species The rhesus monkey, Rhesus macaque has an estimated 8,000 10,000 taste buds within all papillae of the tongue and half of these are within the foliate papillae (Bradley et al. 1985). In the Syrian golden hamster, Mesocricitus auratus there are 230 taste buds within the foliate papillae Rats Rattus norvegicus have an average of 1265 taste buds wi th approximately 455 of those in foliate papillae (Miller and Smith 1984). Within the porcine tongue fungiform papillae have significantly fewer taste buds tha n vallate papillae with an average 3. 9

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68 taste buds per dorsal fungiform papillae and 22. 1 taste bu ds per lateral papillae compared to the 733 taste buds per vallate papillae (Mack et al. 1997). In an aquatic environment, t aste buds are well documented in fish and especially in specialized structures near the mouth of the fish such as barbels (Kotrschal 2000). However, information on taste bud location and density in aquatic mammals is limited. Within semi aquatic species such as those in the suborder Pinnipedia there are fewer taste buds than their terrestrial counterparts and they appear to h ave sensitivit y to sour, bitter, and salty but not sweet (Friedl et al. 1990). Umami the property of a savory or protein rich food, was not tested but p innipeds are thought to be sensitive because of their high protein diet. Pinnipeds are able to detect s alt water gradients within a 4% variance at levels of 30 parts per thousand (Sticken and Dehnhardt 2000). According to Yoshimura et al. (2002) and (2007), the California sea lion, Zalophus californianus californianus and spotted seal, Phoca largha have t aste buds on vallate and fungiform papillae. The walrus Odobenus rosmarus has taste buds few in number (140 or less) but large in size (75 X125 m) on the tongue (Kastelein et al. 1997) This would place the harbor seal with the greatest taste and the California sea lion and grey seal Halichoerus grypus with the least taste (Sonntag 1923). There are no other reports on the number or distribution of taste buds within Pinnipedia. Bottlenose dolphins Tursi ops truncatus have taste buds in pits at the base of their tongue that develop from circumv a llate papillae present in neonates (Kuznetzov 1990). They also have a highly developed gustatory nucleus of the thalamus (Kruger 1959). There are conflicting repor ts on how well bottlenose dolphins can respond to the

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69 four common tastants; salty, sour, sweet and bitter. One study demonstrated dolphins respond at an order of magnitude below human capabilities (Kuznetzov 1990) while another measured a threshold of 0.0 173M for a sour tastant and 1.36X10 5 for bitter, near human thresholds in a 3 5 year old male dolphin (Nachtigal and Hall 1984). Umami has not been tested. Mesoplodon stejnegeri (Sh indo et al. 2008) and striped dolphins, Stenella Coeruleoalba, of varying sex and age (Komatsu and Yamasaki 1980). In a Baiji river dolphin calf, Lipotes vexillifer taste buds were located with fewer found in a young adult female and none in an older adul t female ( Li 1983). These examinations involved small sample sizes and limited comparisons between age and sex. Therefore, the true extent of individual species taste ability and use is difficult to determine currently but in general o dontocetes have the ability to taste and may be using this in food assessment or mate recognition. There are no formal published reports on the use of taste or presence of taste buds in m ysticetes. Within the order Sirenia, the dugong, Dugong dugon has taste buds in pits o f the lateral margins of the tongue (Yamasaki et al. 1980) and the Florida manatee, Trichechus manatus latirostris has taste buds in foliate papillae located on the side of the tongue root (Levin and Pfeiffer 2002 ). In both species it is thought that taste is integral for freshwater detection and food palatability but it is not known if their chemoreceptive use translates to communication of reproductive status (Yamasaki et al. 1980; Reep and Bo nde 2006). In general, s irenian s taste buds are more num erous than cetaceans or pinnipeds (Yamasaki et al. 1980).

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70 The Florida manatee is an endangered species for which increased understanding of their physiology, reproduction and nutrition will help direct management decisions. In manatees, touch is probably the predominant sense for food detection but taste is likely supplementary (Reep and Bonde 2006). Their herbivorous diet increases their vulnerability to toxins which can be detected by a strong bitter taste (Nei 2008) and would indicate a need for good taste detection. In addition, it has long been thought that males use chemoreception to find female manatees in estrus and so it is not known if they use their sense of taste or smell or a combination to do this (Levin and Pfeiffer 2002, Reep and Bonde 200 6). This study sought to characterize the manatee taste buds and compare the cellular components, number and size to other well studied species. In addition, the lingual taste buds and epid ermis Materials and Methods Samples for gross and histological analysis (Table 3 1) were collected from manatees that died within 24 hours of sample collection (i.e., freshly dead) They died from boat strikes, cold stress or natural causes and were broug ht to the state of samples were obtained for collection of tongue roots, portions of the soft palate and from one animal the valve at the base of the nasal passage was avai lable for collection. All samples but one were fixed in 10% Neutral Buffered Formalin (NBF). From one animal three sample cubes, one cm by one cm by two mm, were blocked and fixed in 2% glutaraldehyde 0.1 M phosphate buffered solution for transmission e lectron microscopy (TEM), the rest of the tissue was fixed in 10% NBF.

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71 Table 3 1. Taste bud s amples collected for gross and histological analysis and fixed in 10% NBF TL Total Length and is measured in cm. COD cause of death. *From LPZ102639 three sample s were collected for fixation in 2% glutaraldehyde. Animal Number Sex/TL/COD Sample Collected MEC0853 M/119/Perinatal Tongue Root MSE0943 M/205/Natural Tongue Root MNW0905 M/210/Natural Tongue Root Soft Palate Nasal Valve MSE0909 M/262/Cold Stress Tongue Root Soft Palate SWFTm0820 M/287/Chronic Watercraft Tongue Root Soft Palate LPZ102639 M/309/Chronic Tongue Root Soft Palate MSE0910 F/188/Cold Stress Tongue Root Soft Palate MNW0901 F/212/Cold Stress Tongue Root Soft Palate SWFTm0826 F/261/Chronic Watercraft Tongue Root Soft Palate Gross and Microanatomy The tongue root and oral cavity were examined grossly using a dissecting microscope ( Figure 3 1). Areas of the oral cavity with papillae structures or pits were removed to assess the presence of taste buds. Tongue roots were removed for taste bud counts and measurements. For those tongue roots that were complete, papillae counts were taken over the entire structure Sections of the tongue root containing foliate papillae, soft palate, and nasal valve were taken for histological staining. The samples were embedded in paraffin and sectioned at 5m. The 5m sections were stained with hematoxylin an d eosin (H&E) to outline the general anatomy of the tissue.

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72 Figure 3 1. Midsagittal view of manatee head; T tongue, N.P. n asal p assage, B.C. brain c avity. Note the location of tongue. Taste B ud S ize and Q uantification Tongue roots were removed and if complete, foliate papillae counted ( Figure 3 2). The five micrometer H&E sections were examined and taste bud measurements taken for any taste bud for which the pore was visible. A total of ten manatees had between one and four taste buds with visible pores. This procedure ensured that the measurement was taken at the center of the taste bud and that an individual taste buds was not measured more than once. Two measurements were taken; vertical ly from the t aste pore to the basement membrane and horizontal ly from either end of the taste bud wall at the widest point ( Figure 3 3). The number of taste buds within papillae was calculated For these counts taste buds on either side of the papillae were counted at a magnification of 250X to differentiate taste buds ( Figure 3 4). Some animals had multiple papillae that were counted and multiple sections within the same papillae. Distances between the sections B C N.P. T Dorsal Rostral

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73 were between 10 and 100 m and so there was potential fo r the same taste bud to be counted more than once The average number of taste buds within papillae was calculated from twenty one manatees representing male and females of various ages and causes of death The length of the foliate papillae was measured u sing a micro caliper on the tongue roots of LPZ1027 6 6 and LPZ102939. Figure 3 2. Tongue root location and foliate papillae on side of root. Scale bars represent approximately 1 cm. Fig ure 3 3. Taste bud measurement s. M ade using Leika photographic software at 400X. Both vertical and horizontal measurements were taken. Dorsal Rostral

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74 Figure 3 4. Typical foliate papillae with taste buds 20X and magnification of taste bud at 250X. Note ability to differentiate the four separate taste buds at 250X. Immunohistochemistry A total of twenty were obtained from archived tongue root tissue blocks that had been collected from all freshly dead manatees since 2002 (Table 3 2). Add itionally, those samples collected for examination of taste bud location were used fo staining. Specimens included males and females that died at or near the time of birth, perinatal, as well adult male and female manatees that had var ious causes of death. Sections were mounted on superfrost plus slides and incubated at a dilution of 1:100 overnight with Millipore Anti Universal quick kit followed by an Immpact AEC peroxidase substrate kit. Due to the

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75 large number of specimens, twenty three, the slides were divided into four groups and run separately. Within each group there was a negative control without antibody and a consecutive section from MSW03143 run to control for consistent staining between groups. Staining intensity was assessed by two independent investigators using a blinded analysis on a scale of 0 3 with 0 being no reaction and 3 representing intense reaction Both taste buds and epidermis test. Table 3 2. Samples used for estrogen receptor analysis. TL Total Length and is measured in cm, COD cause of death. Animal Number Sex/TL/COD MSW0830 F/88/Perinatal MSE0630 F/121/Perinatal MEC0470 F/124/Perinatal MSW0601 F/170/Chronic Watercraft MSE0910 F/188/Cold Stress SWFTm0806b F/192/Cold Stress MSW0316 F/198/Cold Stress MEC0616 F/199/Cold Stress MEC0633 F/238/Acute Watercraft LPZ102120 F/259/Chronic Watercraft MSE0504 F/261/Acute Watercraft MSW03143 F/263/Red Tide LPZ102331 F/292/Chronic Watercraft MEC0831 M/95/Perinatal MEC0629 M/97/Perinatal MNE0633 M/116/Perinatal MEC0469 M/120/Perinatal LPZ102331 M/201/Chronic Watercraft SWFTm0802b M/213/Cold Stress MSE0632 M/255/Chronic Watercraft MEC0608 M/276/Unknown LPZ102639 M/309/Chronic Watercraft LPZ102203 M/XXX/Chronic Watercraft

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76 Transmission Electron Microscopy Samples fixed in glutaraldehyde were cut into small portions (2 mm 3 ) and post fixed in 2 % osmium tetroxide for 2 hours at room temperature. They were dehydrated in grades of alcohol and embedded in an epoxy (Epon Araldite) plastic mixture. Ultrathin sections between 70 90 nanometers were sectioned using an ultramicrotome and mounted on a 100 mesh Formvar coated grid. The sections were stained with uranyl microscope (Hitachi H 7000). Results This study confirmed that in the manatee tongue, taste buds are located in the foliate papillae. In addition we found taste buds in one of the seven manatee s examined within the soft palate directly above those located in the tongue ( Figure 3 5 ) and observed what appear ed to be taste buds in the valve at the junction of the nasopharynx and oropharynx ( Figure 3 6 ) The average number of taste buds observed in a single cross section of the foliate papillae was 12 6.4. Within the right tongue root there was an average of 27 4 foliate papillae and in the left side 2 4 4.9 (Table 3 3 ). The average size of the manatee taste buds was measured at 60mX49m (Table 3 4). The average papilla length was 1.13 0 .65mm (Table 3 5). Taking into account the length of the foliate papillae, the width of the taste buds and the number of taste buds observed within a cross section of the papillae, the number of taste buds range d from 77 taste buds/papilla to 520 taste buds/papillae. Using these estimates and the number of papillae within the tongue root, the total estimated number of taste buds within a manatee tongue root was between 3 233 and 31,140.

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7 7 Figure 3 5. Taste buds of the soft palate of LPZ102639 Scale bar equals 100 m. Figure 3 6. Apparent taste buds indicated by arrows, of the nasal valve of MNW0905 Scale bar equals 1 00 m.

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78 Table 3 3 Taste bud and papillae number within manatee tongue roots. TL Total Length and is measured in cm, COD cause of death NM Not Measured Animal Number Animal sex/TL/COD Taste Buds/Papillae Number Papillae Right Number Papillae Left MSW0830 F/88/Perinatal 13 NM NM MEC0831 M/95/perinatal 10 NM NM 24 MEC0629 M/97/Perinatal 10 NM NM 11 MEC0853 M/119/Perinatal 19 NM NM MEC0469 M/120/Perinatal 15 NM NM MSE0630 F/127/perinatal 26 NM NM MSE0910 F/188/Cold Stress 11 NM 22 17 19 18 19 14 19 16 15 9 MSE0909 M/175/cold stress 9 28 26 9 8 7 10 11 SWFTm0806b F/192/Cold Stress 14 NM NM 11 MSW03176 F/198/Cold Stress 25 NM NM 11 LPZ102106 M/201/Chronic Watercraft 9 NM NM MSE0943 M/205/Cold Stress 7 21 17 5 MNW0905 M/210/Natural 6 NM NM 9 MNW0901 F/212/Cold Stress NM NM 18 SWFTm0802b M/213/Cold Stress 18 NM NM MEC0633 F/238/Acute 3 NM NM 13 MSW0696 M/251/natural 17 NM NM MSE0632 M/255/Chronic Watercraft 7 NM NM 12

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79 Table 3 3 Continued Animal Number Animal sex/TL/COD Taste Buds/Papillae Number Papillae Right Number Papillae Left SWFTm0826 F/261/Cold Stress 6 NM NM 7 5 4 6 4 9 3 6 MSW03143 F/263/red tide 34 NM NM LPZ102765 F/267/Chronic Watercraft NM 29 24 LPZ102941 F/272/Chronic Watercraft NM 32 29 MEC0680 M/276/Unknown 9 NM NM MSW0332 M/307/Red Tide 12 NM NM LPZ102939 NM 27 29 Average S.D. 126.4 274 244.9 Table 3 4 Taste bud size within manatee tongue roots. TL Total Length and is measured in cm, COD cause of death. Animal Number Animal sex/TL/COD Taste bud vertical size (m) Taste Bud Horizontal Size (m) SWFTm0802b M/213/Cold Stress 72 71 MEC0633 F/238/Acute 87 67 MEC0629 M/97/Perinatal 45 31 MSW03176 F/198/Cold Stress 73 51 MEC0831 M/95/Perinatal 64 38 41 33 MSE0630 F/127/Perinatal 63 39 37 40 38 26 SWFTm0826 F/261/Cold Stress 84 58 93 55 73 30 55 55 MSW0696 M/251/Natural 61 73 70 60 MEC0853 M/119/Perinatal 57 53 47 58 58 76 44 47 MSW0332 M/307/Red Tide 44 40 66 44 42 40 Average S.D. 6016 4914

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80 Table 3 5. Measured length of foliate papillae from the tongue roots of two manatees. The overall average length was 1.13 0 .65mm Foliate Papillae LPZ102939 LPZ102765 Left side Right side Left Side Right Side 1 1 0.5 0.5 1 2 0.5 1 0.5 2 3 0.5 1 0.5 0.5 4 0.5 1 1 1.5 5 1 0.5 1 1 6 1.5 0.5 1.5 1.5 7 1 0.5 2 1 8 1 1 1.5 1 9 0.5 0.5 0.5 1 10 0.5 0.5 1 0.5 11 0.5 1 1 2 12 0.5 2 1 0.5 13 0.5 2 0.5 1 14 0.5 1 2.5 1 15 0.5 1.5 2.5 1.5 16 0.5 2 3 1.5 17 0.5 2 1 1.5 18 1 1 4 2 19 1 1.5 1 2 20 0.5 1 1 1.5 21 0.5 0.5 1 0.5 22 1 2 1 0.5 23 1 1.5 2.5 0.5 24 1 1 1 1 25 1 2 NA 1 26 1 3 NA 1.5 17 1 0.5 NA 1 28 1.5 NA 1 29 1 NA NA 2 Average 0.79 1.20 1.38 1.19 of these receptors within the epidermis and taste buds of the manatee ( Figure 3 5). There was a significantly greater amount of reaction in epidermis of the tongue root

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81 compared to the taste buds themselves ( p = 0 .0007 test) Therefore, the comparisons have been split into epidermis and taste bud groups (Table s 3 6 and 3 7 ). Figure 3 7 staining within the lingual root of an adult manatees. A) taste buds and B) epidermis of an adult male manatee, MEC0608 and C) taste buds and D) epidermis of an adult female MSW03143. A D C B

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82 Table 3 6. Amount of estrogen receptor reaction, average of two within the epidermis of the Florida manatee tongue root. TL Total Length and is measured in cm, COD cause of death. Animal Number Sex/TL/COD Nuclear Staining Y/N Amount of Stain (0 3) MSW0830 F/88/Perinatal N 1.5 MSE0630 F/121/Perinatal Y 2.5 MEC0470 F/124/Perinatal N 1.5 MSW0601 F/170/Chronic Watercraft N 2 MSE0910 F/188/Cold Stress Y 2 SWFTm0806b F/192/Cold Stress Y 2 MSW0316 F/198/Cold Stress N 1.5 MEC0616 F/199/Cold Stress Y 3 MEC0633 F/238/Acute Watercraft Y 3 LPZ102120 F/259/Chronic Watercraft N 1 MSE0504 F/261/Acute Watercraft Y 2 MSW03143 F/263/Red Tide Y 3 LPZ102331 F/292/Chronic Watercraft Y 2.5 MEC0629 M/97/Perinatal N 3 MNE0633 M/116/Perinatal Y 1.5 MEC0469 M/120/Perinatal Y 2 LPZ102331 M/201/Chronic Watercraft N 1.5 SWFTm0802b M/213/Cold Stress Y 1 MSE0632 M/255/Chronic Watercraft N 1 MEC0608 M/276/Unknown N 1.5 LPZ102639 M/309/Chronic Watercraft Y 3 MEC0831 M/95/Perinatal N 2 LPZ102203 M/XXX/Chronic Watercraft Y 1.5 All manatees had estrogen in the cytoplasm but only 17 out of 23 manatees examined had some sort of nuclear staining; nine of which had staining in the taste buds. There was no variation in age, sex or COD as to whether th ere was or wasn't nuclear staining. Five of the manatees had nuclear staining in both epidermis and taste buds, eight had staining in epidermis only, and four had staining in taste buds only. Perinatal taste buds were significantly more stained in males th an females ( p = 0.015, but almost exactly the same in adults (p = 1

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83 test) The epidermis of adult females were more strongly stained (2.2) than males (1.8) but not significantly (p = 0.12 test) Table 3 7 Amount of estrogen receptor beta reaction ratings, within the taste buds of the Florida manatee. TL Total Length and is measured in cm, COD cause of death. Animal Number Sex/TL/COD Nuclear Staining Y/N Amount of Stain (0 3) MS E0630 F/121/Perinatal N 0.5 MSW0601 F/170/Chronic Watercraft N 1 MSE0910 F/188/Cold Stress N 0.5 SWFTm0806b F/192/Cold Stress N 0.5 MSW0316 F/198/Cold Stress Y 1.5 MEC0616 F/199/Cold Stress Y 1.5 MEC0633 F/238/Acute Watercraft N 1.5 LPZ102120 F/259/Chronic Watercraft N 0.5 MSE0504 F/261/Acute Watercraft N 2 MSW03143 F/263/Red Tide Y 2 LPZ102331 F/292/Chronic Watercraft Y 1.5 MSW0830 F/88/Perinatal N 0.5 MNE0633 M/116/Perinatal Y 1.5 MEC0469 M/120/Perinatal N 1.5 LPZ102331 M/201/Chronic Watercraft Y 1.5 SWFTm0802b M/213/Cold Stress Y 0.5 MSE0632 M/255/Chronic Watercraft N 0.5 MEC0608 M/276/Unknown Y 1.5 LPZ102639 M/309/Chronic Watercraft N 2 MEC0831 M/95/Perinatal Y 1.5 MEC0629 M/97/Perinatal N 2.5 LPZ102203 M/XXX/Chronic Watercraft N 1.5 Ultrastructure Examination by transmission electron microscopy demonstrated the location of the taste pore and basement membrane as well as the four types of taste cells : I, II, III, and IV. Type I contain a darker nucleus and cytoplasm while type II has the lighter nucleus and cytoplasm. Type III can be identified by their dark granules and type IV were found near the basement membrane ( Figure 3 8 ).

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84 Figure 3 8 Electron micrographs of manatee taste buds. A ) The beginning of the taste pore of the bud, Taste cell I, II and taste pore (TP), scale bar equals 10m. B ) t he base of a taste bud including basement membrane (BM) and each of the four taste cells; I, II, III IV, scale bar=20 m. Discussion The estimate of between 77 and 520 taste buds/papillae falls under what has been observed in other species circumvallate papillae, which are similar in structure and location to foliate papillae (Davies et al. 1979, Mack et al. 1997). On the tongue of the bovine, Bos t aurus the number of taste buds per papillae had extreme variation. Some papillae had fewer than 200 taste buds/papilla and one papilla had 1,786 taste buds with an average of approximately 800 taste buds/pap illa Porcine tongues had between 583 875 taste buds/ circumvallate papilla (Mack 1997). The large variation of taste bud estimate between 3,233 and 31,140 is due to the small sample size and limited examination of any single animal. Future experiments shou ld focus on a more standardized examination of each tongue root and including histological sectioning throughout the length of the papillae, such as in the examination of the porcine tongue (Mack et al. 1997). The actual number of taste buds within the IV I A I II II III TP II I BM

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85 man atee tongue is likely between the above estimates which would put it at a similar estimate as t he bovine tongue which has between 14,765 and 21,691 taste buds (Davies et al. 1979). Another animal with a similar range is the rhesus monkey with estimated 8,0 00 10,000 taste buds (Bradley et al. 1985). The manatee has far more taste buds than the Syrian golden hamster, Mesocricitus auratus at 723 taste buds have an average of 1 265 taste buds (Miller and Smith 1984). Although exact counts are not given, t he manatee appears to h ave more taste buds per papilla than the hippopotamus Hippopotamus amphibius amphibius ( Yoshimura et al. 2009) and hyrax Procavia capensis (Yoshimura et al. 2008). The concentration of taste buds within the foliate papillae of the tongue root may be correlate d to food mastication and the transportation of the resultant mixture of water and food particles. There are narrow grooves on either side of the manatee s tongu e that lead from the palate to the root of the tongue where taste buds are located ( Figure 3 9 ). This anatomical design would allow for the mixture to be tasted before the bolus was trans ported to the back of the mouth. It would also allow for quicker expu lsion of non desired food material. The position of taste buds within the foliate papillae may correlate with the herbivorous diet of the manatee. In humans there is a strong response to the highly bitter compound quinine hydrochloride on the vallate pap illae, which are comparable to foliate, at the base of the tongue as compared to the fungiform papillae on the front of the tongue (Collings 1974). It is likely that similar taste receptors are located in the foliate papillae at the base of the tongue of h yrax and manatee as these are herbivorous

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86 species. Through b ehavioral studies examining manatees sense of bitter taste as well immunohistochemical staining for these receptors within t he tissue confirm ation of this hypothesis would be made Figure 3 9. Mid sagittal section of a manatee head with a transverse section demonstrating the groove between tongue and teeth (arrow) T tongue, NP nasal passage, BC brain cavity TR tongue root The iso lation of taste buds in manatee s foliate papillae may be explained by their habitat and diet. Differences in taste bud number a nd location between two similar families of fish, the B lennid ae and G obiid ae indicate that differing foraging strategies and diet can affect chemosensory structure distribution (Fishelson and Delarea 2004) Both fish families have similar body types and l ive in the same ecological niche of coral reefs but have differing diets. An examination of the taste buds on the lips and mouth of these fish demonstrated differences. In gobies the number of taste buds increased with Dorsal BC NP T NP T Dorsal R o st ra l R o st ra l C a u d al T R

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87 age and therefore growth rate, reachi ng a total of 7500 taste buds. These were situated mostly on the lips and towards the front of the oral cavity. B lennies were observed to have many fewer taste buds, no more than 1000, with most of them located on the posterior part of the mouth. The amoun t and distribution of these taste buds correlate d were determined to be much less selective than gobies and gobies fed predominantly by suctioning in their prey from bottom sediment and so depend on taste instead o f visual cues (Fishelson and Delarea 2004). These results indicate that among fish and perhaps all species there is a correlation between diet type feeding strategy and the use of taste. Quantification of soft palate taste buds of the manatee is impractical due to their scarcity and difficult y to locate. Taste buds located on areas other than the tongue have had limited study and there is much debate on what their purpose. A current hypothesis that has received the most attention has been that epi glottal taste buds, which would correlate with those found on the manatees valve, aid in the breathing response to ensure that animals do not aspirate liquid or food particles (Bradley et al. 1980). This hypothesis is supported by behavioral data in which newborn piglets and lambs were subjected to water and milk placement on their laryngeal surface. Their breathing was monitored and it was found that they went through an apneic period that persisted to death if allowed (Downing and Lee 1975). It is also hypothesized that soft palate taste buds aid in nipple attachment in young animals (El Sharaby et al. 2001) and provide supplementary taste information in all animals (Miller and Spangler 1982). Therefore, soft palate taste buds may be more prevalent in yo unger manatees.

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88 The cellular structure of the taste buds in manatees is similar to other species. Each cell type is represented. The size of manatee taste buds is comparable to other mammals (Sonntag 1923) They are larger than both young and adult rats, 46m, (Mistretta and Baum 1984) but smaller than the walrus 75 X125 m (Kastelein et al. 1997). The size of the taste bud however, does not necessarily indicate the amount of taste transduction. The number and distribution of cells within individual taste buds are more indicative of taste ability. When rat and mouse taste buds were compared it was found that the rat taste bud is larger in volume than the mouse but the mouse has a significantly larger density of taste cells within the taste bud (Ma et al. 20 07). Therefore, the sense of taste is not solely dependent on how many taste buds there are but how many cells are present to detect chemicals. A similar calculation of manatee taste bud density and taste cell number would allow for a comparison with the t raditional laboratory species of rat and mouse. manatee in all ages and sexes indicates that it has a universal purpose and has an important function within the oral cavity. The absence of nuclear staining in some animals may have been a consequence of inadequate fixation. The samples were collected over a series of years by various people and although a protocol was in place for limited fixation followed by immediate embedding, this may not have always been the case. The significant difference in perinatal taste bud positive reaction between males and females requires more sample s to verify. The epidermis of adult females reacted more positively (2.2) than that of adult males (1. 8) but not significantly (p= 0.12).

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89 It has been documented that estrogen is an important hormone in the production of mucous and saliva, although the exact pathways are not known (Valimaa et al. 2004). Not only is the production of mucous and saliva import ant to the transduction of taste but estrogen itself may be a component of taste transduction (Toyoshima et al. 2007). Although in an aquatic environment the manatee would not be as reliant on saliva to transport chemicals, the mucous would be important to maintain chemicals within the region of taste buds for an adequate amount of time to be sensed without being cleared away by external water. This likely also explains the location of taste buds at the interior of the buccal cavity. Any taste buds closer t o the front of the mouth would be more susceptible to clearing by water. Another potential function for estrogen receptors within the mouth of the manatee is for the reproductive communication. Within the goldfish it is well documented that males respond s trongly to varying levels of 17 estradiol ( Bjerselius et al. 2001). This is Manatee hormones vary with reproductive cycling similarly to other species and they may have evolv ed the ability to detect these variations in chemicals similar to the goldfish (Sorenson and Stacey 1999). Regardless of the function(s) for estrogen receptors in the mouth of the manatee there is concern about chemicals that may mimic estrogen in the environment. The effect s or causing an unnecessary increase in their function. Estrogen receptors are of special concern in reference to mimicking chemicals and although all animals are sensitive to estrogens, those that reside in an aquatic setting are especially vulnerable.

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90 Al though not demonstrated in aquatic mammals, there are several examples of aquatic vertebrates and invertebrates that are affected physiologically and behaviorally by estrogen mimicking compounds (Cheek et al. 1998). Biodegradation products of detergents ha ve direct interaction with fish estrogen receptors (White et al. 1994) and when exposed, male fish have developed significantly higher levels of vitellogenin, an egg yolk protein (Purdom et al. 1994). Also, polychlorinated biphenyls (PCBs) have been demons trated to have feminizing effects on developing male red eared sliders in the same way as exposure to estradiol (Bergeron et al. 1994). Additional chemicals that have been implicated in disruption of estrogen receptors include phytoestrogens, bisphenolics (from plastic degradation) and organochlorine pesticides (Shanle and Xu 2011). The distribution and amount of taste buds within the oral cavity of the manatee indicate that the sense of taste is important to the manatee. The presence of estrogen is also interesting and with further study may increase the understanding of the use of estrogen as a chemical signal in mammals in general. R esearch is also environment on the sense of taste.

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91 CHAPTER 4 THE NASAL CAVITY AND OLFACTORY EPITHELIUM OF THE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS Background The nasal cavity of mammals is the site of temperature and humidity control, filtration and chemical detection of inspired air. The epithelium : squamous, respiratory and olfactory is responsible for these functions. S quamous epithelium is found in the rostral most nasal passage and protects the other epithelium from large obstructions and in many species contains hai r as an added filter. The respiratory epithelium has nasal associated lymphatic tissue to support immune response, and mucous producing goblet cells that facilitate warming of the air, control of humidity and removal of inhaled particulate material The o lfactory ep i thelium is typically located in the dorsoc auda l portion of the turbinates and is composed of receptors that project their signals through the basement membrane to form cranial nerve one ( Gross et al 1982 ). M ammals have a series of turbinates that originate from various bones including the ethmoid, nasal and maxillary. These turbinates serve to increase the surface area within the nasal cavity to provide access to the epithelium for temperature control, humidity control and chemical detection. The maxillary and nasal turbinates have been associated with respiratory epithelium while the ethmoturbinates are where olfactory epithelium can be found (Kavoi et al. 2010, Stobel et al. 2010, Van Valkenburgh et al. 2011). Of the various functions of the nasal cavity, olfaction is the most variable between species with regard to the amount and location of olfactory epithelium Variation occurs for several reasons including habitat, diet and use of chemical communication. For example, the dog, Canis familia ris a carnivore, has a great density

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92 of olfactory receptors and a thicker olfactory epithelium than sheep, Ovis aries an herbivore (Kavoi et al 2010). Domestic dogs have a wide variation in the surface area of olfactory epithelium, 169.46 cm 2 in the German Shepherd compared to 26.89 cm 2 in the Pekingese However, this is much higher than humans 10 12.5cm 2 and rabbits 7.4 10.2 cm 2 (Adams 1972). The most extreme example of variation with habitat is within the o dontocetes ( Oelschlager and Buhl 1 985 ), fully aquatic mammals which do not have olfactory capabilities Other fully aquatic mammals; the m ysticet es ( Oelschlager 1989 ) and s irenians (Mackay Sim et al 1985) do appear to maintain olfactory capabilities. The Odontocet i (also known as toothed whales) that have been examined do not have an accessory and main olfactory bulb, olfactory tract or anterior olfactory nucleus as adults (Oelschlager and Buhl 1985, Marino 2001, Oelschlager et al. 2008a). It appears that the bulb m ay have been present in early evolutionary lineages of the delphinids and is lost in present day species during embryonic development (Oelschlager and Buhl 1985). In addition to the regression of the olfactory neuroanatomy, a histological study characteriz ing the nasal complex of fifteen harbor porpoises, Phocoena phocoena found no evidence of olfactory epithelium (Prahl et al. 2009). There are no published studies on the behavioral ability of odontocetes to react to olfactory cues. In addition to the anat omic al evidence, the investigation of the o dontocete genome for olfactory receptor genes corroborates gradual evolutionary los s of olfaction (McGowen et al. 2008) Several independent pseudogenization events support the theory of the loss of an olfactory s ense over evolutionary time. This genomic and

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93 anatomic evidence in addition to the lack of behavioral accounts of o dontocetes using olfaction indicates that between the Miocene and present day these species lost the ability to smell. This is invariably due to the lifestyle and environment of these animals in addition to the energetic cost required to maintain an olfactory system. Within th e baleen whales, Mysticeti Oelschlager (1992) examined embryos and early fetuses from three different species : blue, Ba laenoptera musculus fin, Balaenoptera physalus and humpback, Megaptera novaeangliae whale s Unlike Odontocetes the olfactory bulb, tract, and epithelium of the Mysticetes did not regress and was well formed and evident in a 105mm crown rump length ( CRL ) fin whale fetus (Oelschlager 1992 ). In addition to the embryologic evidence, of adult brains from 11 b owhead whales, Balaena mysticetus (Duffield et al. 1992) and a single h umpback (Breathnach 1955) contained olfactory peduncl es, nerve fibers and small holes in the cribiform plate of the ethmoid bone which would allow the olfactory nerve to pass into the brain. In a recent study the b owhead whale olfactory bulb was located and measure d 0.13% of the brain weight and 51% of the olfactory receptor genes were intact (Thewissen et al. 2011) Anecdotal behavioral evidence and a highly reduced but intact olfactory system indicate that m ysticetes retain some sense of airborne smell (Oelschlager 1989). Baleen whales spend a majority of their time at or ne ar the surface to feed, unlike many of their toothed counterparts that dive deep to locate prey (Berta et al. 2006). Therefore the use of airborne scents to locate groups of zooplankton or other prey as well as potentially locate signals given off by pred ators or mates would correspond with the anatomic evidence of the m ysticetes. It would be interesting to see if there is a variation

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94 in olfactory size between those m ysticetes that feed near the surface such as the bowhead and those that feed on inverteb rates from botto m sediment like the gray whale Eschrichtius robustus Within the order Sirenia, t he Florida manatee, Trichechus manatus latirostris is a fully aquatic mammal that inhabits marine and freshwater areas and is a n herbivore. The manatee exhibits the behavior of sniffing in captivity Unlike o dontocetes but similar to m ysticetes, the manatee possesses a small olfactory bulb (Wiseman and Reep Pers. Comm., Mackay Sim et al 1985, Reep et al. 2007 ). Although the manatee does not have a vomeronasal organ (VNO) it does have what appears to be olfactory epithelium (MacKay Sim et al. 1985). N o further study has been reported on manatee olfaction This study seeks to characterize the manatee olfactory epithelium and turbinate system and compare this to other mammals. It is expected that the manatee does possess olfactory epithelium as indicated by Mackay Sim et al. (1985) but that there is less than observed in terrestrial mammals. Materials and Methods Samples were collected from manatees that that died wi thin 24 hours of sample collection (i.e., freshly dead) They were killed by b oat strikes, cold stress or natural Laboratory in St. Petersburg, FL. From eight animals; e ither the entire head or half of a m id s agittal cut of the head were collected and fixed in 10% N eutral Buffered Formalin (NBF) for 24 48 hours (Table 4 1). The head was then cut on a bandsaw into approximately 1 .5 cm thick sections along either the transverse or horizontal plane ( Figure 4 1). The sections of nasal cavity and turbinates were then decalcified for five to

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95 seven days in Cal Ex decalcifying solution and cut into smaller samples approximately two square centimeters for placement in histology cassettes. Figure 4 1. Head of a male manatee, MNW0905, used for nasal examination. C ut in the A) transverse plane right side, and B) horizontal plane left side Table 4 1 Samples collected for nasal cavity and olfactory epithelium evaluation TL Total Length and is measured in cm, COD cause of death. Animal Number Sex/TL/COD /Age Class Plane of Section/Side of Head MSE0910 F/188/Cold Stress /Calf Transverse/Left MNW0901 F/212/Cold Stress /Calf Transverse/Right Transverse/Left SWFTm0826 F/261/Chronic Watercraft /Adult Transverse/Right Transverse/Left MEC0853 M/119/Perinatal /Newborn Calf Transverse/Left Transverse/Right MNW0905 M/210/Natural /Calf Horizontal /Right Transverse/Left MSE0909 M/262/Cold Stress /Adult Horizontal /Right Horizontal /Left SWFTm0820 M/287/Chronic Watercraft /Adult Transverse/Left Transverse/right LPZ102639 M/309/Chronic /Adult Transverse/Left From one animal, an adult female (LPZ102765) that was euthanized after complications from a chronic boat strike, the entire head was perfused with 4% paraformaldehyde and the brain collected for another study. The head was then cut at 2 midline al ong the sagittal plane to expose the lateral portion of the nasal cavity B A Rostral Dorsal Caudal

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96 and turbinates. The head was post fixed in 10% NBF for 18 hours and nasal and olfactory epithelium collected for histological assessment (Figure 4 2). Figure 4 2 Section of manatee head, LPZ102765, from which olfactory and nasal samples were collected. Light Mi croscopy S amples were embedded in paraffin and sectioned with a rotary microtome at 5m. The 5m sections were stained with hematoxylin and eosin (H&E) to outline the general anatomy of the tissue. A few samples were stained with periodic acid Schiff (PAS) to stain glycogen rich material and identify mucous rich secretions and their secreting cells Each stained slide was examined and compared to the loca tion of the nasal passage from which it was collected to identify the location of olfactory epithelium. When possible the olfactory epithelial thickness was measured from basal lamina to apical surface using the Leica Application suite v. 3.5.0 digital mic rometer ( Figure 4 3) Caudal Dorsal

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97 Figure 4 3. Measurement of olfactory thickness. Immunohistochemistry From the female manatee LPZ102765 five m s ections were mounted on superfrost plus slides and incubated at a dilution of 1:10 0 0 for one hour with Wako Olfactory Marker Protein (OMP) antibody using a Vector Vectastain Universal quick kit followed by an Imm PACT AEC peroxidase substrate kit. The location and density of olfactory epithelium was compared to the areas within the nasal passage from which the samples wer e collected. Results Squamous epithelium lined the most rostral area of the nasal cavity with occasional vibrissae for the first few centimeters of the canals. Respiratory epithelium began midway between section 1 and 2 on Figure 4 4 and continued along the nasal

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98 passage and both the middle and dorsal nasal turbinate s It included a majority of goblet and mucous producing glands as well as sections of large veins. The olfactory epithelium was found throughout the ethmoturbinates and bordering septum Grossly, i t is a dark yellow, brown color that can be differentiated by both color and texture from the lighter and thicker respiratory epithelium For identification of turbinate structure the transverse sections were most elucidative ( Figures 4 4 and 4 5 ). The horizontal sections helped to identify the type of epithelium within the turbinates. Figure 4 4 Manatee head with sections dividing the general area of bandsaw cuts made on a perinatal male, MEC0853. 1 2 3 4 5 6

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99 Figure 4 5 Cross sections of the nasal cavity of a perinatal male Florida manatee, MEC0853 Each transverse section correlates to a segment from Figure 4 4 A) section 1, B) section 2, C) section 3, D) section 4, E) section 5, and F) section 6. Ethmoturbinates 1 3 where ol factory epithelium can be found and the ( M ) Middle nasal turbinate and ( D ) dorsal n asal turbinate that contain respiratory epithelium Scal e bar approximates one centimeter. 1 D 2 D 1 2 3 D M A F C B D E M

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100 Light M icroscopy Throughout section one and a portion of section two of Figure 4 4 the epithelium progressed from squamous to ciliated columnar with extensive mucous glands and goblet cells ( Figure 4 6 ) A majority of the epithelium in the ventral portion of sections two, three and four was heavily embedded with goblet cells a nd mucous glands like those in Figures 4 7 and 4 8 Within the ethmoturbinates of sections five and six olfactory epithelium was predominant ( Fig ures 4 9, 4 10) and found in a small section of the caudal most portion of the dorsal nasal turbinate ( Figure 4 11 ). The average thickness of the olfactory epithelium was 54.79 m (Table 4 2) Sample size was too low to determine any statistical variation with animal sex or age. Table 4 2. The olfactory thickness of various manatees from different areas of the olfactory system as represented by different block numbers. Animal ID tissue block number Olfactory Thickness (m) LPZ102765 21 77.355 LPZ102765 22 60.59 LPZ102765 23 48.84 LPZ102765 24 43.88 LPZ102639 23 44.58 MSE0909 48 61.895 MSE0909 42 58.79 SWFTm0826 37 56.655 MEC0853 30 40.565 Average 54.79 Figure 4 6 C ross section of mucous producing cells from adult female, MNW0901 H&E 20X Taken at approximately section 3 of Figure 4 4

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101 F igure 4 7 Mucous glands of adult female, MNW0901 at approximately section 2 of Figure 4 4 A ) 400X H&E and B) 250X, PAS. Figure 4 8 Respiratory epithelium within the nasal cavity of the Florida manatee. A) perinatal male, MEC0853, at approximately section 3 of Figure 4 4 at 250X with staining of goblet cells (G) using PAS and B) an H&E of an adult male, SWFTm0820, at approximately sect ion 2 of Figure 4 4 with veins ( V) and Nasal Glands (NG), scale bar = 500m Figure 4 9 C ross section from adult female, MNW0901 demonstrating olfactory epithelium H&E, 100X approximately section 6 of Figure 4 4 A B A B V NG G

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102 Figure 4 10 Section of manatee head along sagittal plane and corresponding epithelial lining within Ethmoturbinates II and III and dorsal nasal turbinate ( D ) All stained with H&E Squamous Epithelium (SE), Columnar Epithelium (CE), Goblet Cells (G) Vein ( V) ; A) Olfactory epith e lium at 250X, B) Olfactory epithelium at 100X, C) Transiti onal epithelium with scale bar 100 m D) Respiratory epithelium at 250X. Caudal Dorsal II III R A D C B BG BG OE OE SE eE CE eE G eE V eE

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103 Figure 4 11 Diagram representing the turbinate system and location of epithelial types within the manatee head, T=tongue. The middle nasal turbinate (M) is a small extension of bone at the cranial part of the nasal cavity while the dorsal nasal turbinate (D) extends across the entire dorsal portion of the cavity. Ethmoid turbinates (I, II, and III) as well as a small portion of the caudal na sal turbinate are where olfactory epithelium can be found. Immunohistochemistry Localization of OMP was successful but not consistent. Only one block of epithelium identified as olfactory with H&E reacted to the OMP antibody but both the receptors and neu rons stained darkly ( Figure 4 1 2 ). There was excessive background stain. T he process degraded the tissue and caused breaks and tears that may have impaired the ability to see the stain within some samples

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104 Figure 4 1 2 OMP s taining of olfactory epithelium from an adult female, LPZ102765. A) Olfactory epithelium and nerve fibers with scale bar 100 m, B ) and C) Olfactory receptors scale bar at 50 m, D) nerve bundle stained with OMP and scale bar of 100 m. Discussion Manatees have far fewer and larger turbinates than found in animals with a highly developed sense of smell, macrosm a tic such as cats, dogs and bears but a similar turbinate st ructure as observed in microosma tic species with a poor sense of smell such as humans a nd rabbits (Adams 1972) Unlike other examined aquatic mammals such as p inniped s the manatee does not have highly developed maxilloturbinates which are used for heat and gas exchange (Van Valkenburgh et al. 2011). This is likely because manatees live in a warm water environment and do not dive deep. However, the manatee does have extremely large veins which increase the A B C D

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105 amount of blood present for heat exchange. The large venous system likely acts as a thermoregulatory system depending on the need of the animal Also the dense mucous producing glands, including goblet cells, are another factor that would enhance gas exchange within the nasal cavity. Other marine mammals likely have similar morphology within the venous system of the lamina propria of the respiratory epithelium. The turbinate structure of the manatee is most similar to the hyrax its closest evolutionary relative. Within the ethmoid region, its three turbinates are in a similar position to those of the hyrax. The hyrax does have a vom eronasal organ (VNO) (Stobel et al 2010), which the manatee lacks (Mackay Sim et al. 1985). Of the other close evolutionary species to the manatee t he elephant has five and the aardvark nine ethmoid turbinates that are especially convoluted compared to t he manatee and hyrax The elephant and aardvark also have a VNO As discussed by Stobel et al (2010) the structure of the ethmoid region is not indicative of placement within evolutionary systematics. However, the structure does support that the manatee a nd hyrax are mi croosma tic compared to the macr osm a tic elephant and aardvark (Stobel et al. 2010) The location of olfactory epithelium within the ethmoturbinates supports what was observed by Mackay Sim et al. (1985). The manatee olfactory epithelium does react to O MP, although not consistently. This inconsistency may be due to the fixation process, tissue preparation or may reflect a variation in OMP expression within the manatee. Future experiments should use optimal preservation for IHC and include mult iple animals to verify the antibody binding. The antibody does bind to the olfactory neurons and therefore could be used in the brain to verify regions of olfactory transduction. The density of olfactory receptors reacting to the OMP is less than other spe cies as is the

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106 thickness, 54.71 m compared to dogs, 72.5 m and sheep 56.8 m. The greatest length of ethmoturbinate, ~3 cm is less than both sheep ~3.4 cm and dog ~4.3 cm (Kavoi et al. 2010). Future research should calculate the area that is covered by olfactory epithelium to compare to what is known about other vertebrate species. The best methods would take advantage of digital systems that allow for accurate measurements of structures. As expected, the manatee has less olfactory epithelium than most terrestrial m ammals, including sheep, dogs, and humans. H owever there are still substantial amounts of receptors especially considering the decreased amount of exposure time to odorants Therefore, the question remains of why manatees have maintained the ir ability to smell while other fully aquatic species, o dontocetes, have not. The manatee does masticate food at the surface occasionally which would allow signals to be transported to the olfactory receptors Although not a social species, the manatee m aintain s strong mother/calf bonds for the first few years of life and forms large mating herds ( Hartman 1979 ) The mother and calf will breathe simultaneously and this likely helps form a bond. Manatees in a mating herd spend more ti me at the surface than during other activities which would suggest communication through olfactory signals. Also, during cold winter months, manatees congregate within warm water refuges. They breathe in unison while thermoregulating and this would present an opportunity to smell each other I t is unlikely that the manatee transports chemicals to the olfactory epithelium underwater because of the valve that separates the nasal passage from the orophary nx. However, it is possible that retronasal smelling could occur similar to how terrestrial mammals perceive flavor.

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107 To determine how well the manatee is able to detect odorants and to determine whether retronasal exposure of odorants is possible behavio ral experiments would be the most clarifying. Go/no go trials have been completed with captive manatees examining their ability to see, hear and touch ( Reep and Bonde 2006 ). A similar study design using odorants typical of the manatee environment and app lied to both the nares and mouth would begin to reveal the extent of olfactory ability. In addition to behavior experiments, e receptor genes c ould indicate the range of odor ants they are able to detect. Genetic data in dicate that there are 400 1000 different olfactory receptors in mammals (Parmentier et al 1992, Ressler et al. 1994) while only 50 100 exist in fish (Ngai et al. 1993, Barth et al. 1996). There are currently two known receptor classes characterized type I and type II that evolved from common ancestral genes. The absence of class I receptors in terrestrial mammals and birds and the existence of t hese receptors in fish indicate that these are responsible for detection of water soluble odorants. Type II rec eptors are found in terrestrial species and so are thought to be specialized for volatile odorant detection. In addition nonfunctional pseudogenes of type II receptors are found in the striped dolphin, Stenella coeruleoalba, genome which correlates with the loss of olfaction as dolphins entered an aquatic environment (Freitag et al. 1998). are actively producing RNA. One would expect manatees to have type II receptors and a similar olfactory receptor gene repertoire as the bowhead whale in which 51% of the olfactory receptor genes are intact (Thewissen et al 2011). This would imply that the manatee is able to detect a smaller number of odorants than terrestrial mammals bu t of

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108 those odorants they detect the concentrations needed would likely be the same or less than terrestrial mammals as indicated by the amount of epithelium present. These data indicate that the manatee is capable of smelling. For what purpose the manatee retains a sense of smell is yet to be determined. Through additional examination of the manatee genome and behavior the extent of olfactory use can be ascertained

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109 CHAPTER 5 MALE FLORIDA MANATEE, T RICHECHUS MANATUS LATRIROSTIS BEHAVIORAL RESPONSE TO FEMALE MANATEE URINE FROM DIFFERENT REPRODUCTIVE TIMEPOINTS Background Reproductive chemical signaling is used by many vertebrate species ranging from large terrestrial elephants Loxodonta africana to small aquatic goldfish Carassi us auratus auratus Terrestrial mammals including the elephant use highly volatile compoun ds as pheromones (Rasmussen 1999 ) ; whereas in an aquatic environment a chemical signal is typically soluble and of a size small enough to be dispersed (Wyatt 2006). The use of chemical cues by fully aquatic mammals has never been verified although numerous anecdotal accounts exist (Vosseler 1924, Hartman 1979, Lowell and Flanigan 1980). Mammals use a wide variety of chemical signals to broadcast their reproductive status to the same and opposite sex. These signals can both attract and deter potential mates depending on the age and condition of the animals involved. Chemicals are energetically cheap to produce and broadcast which is why they are most likely ubiquitous among species for the transmission of reproductive signals (Bigiani et al 2005). Very few mammal species have been studied in depth for their use of sexual pheromonal cues. Laboratory species including the mouse, Mus musculus rat Rattus norvegicus and hamster, Mesocricetus auratus have been examined in depth while the elephant is the predominant example of the wild use of pheromona l cues by a mammal. These species use a combination of excreted signals in urine and glandular status to females and other males through a combination of chemicals commonly

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110 known as musth. These excretions contain many chemicals including the pheromones frontalin and acetones (Schulte and Rasmussen 1999). Both of these represent the typical volatile odorants used as a pheromone by terrestrial species and in fact a specific ac etone, (Z) 7 dodecen l yl acetate is used by both moths and elephants to express reproductive receptivity (Rasmussen et al. 1996). In non mammalian aquatic species it has been found that two types of signals are used, either steroid derivatives as with the goldfish (Dulka et al. 1987), or peptide pheromones as with the newt Cynops pyrrhogaster (Kikuyama et al. 1995). The Florida manatee, Trichechus manatus latirostris is a close evolutionary relative to the elephant but its reproductive behavior is not w ell understood due to the difficult nature of studying a sparsely populated endangered species in a low visibility, aquatic environment. In addition, the manatee has limited social interactions These include mother/calf bonds that last between two and thr ee years (Hartman 1979) as well as mating herds in which many males follow a receptive female for up to four weeks ( Rathbun et al 1995, Larkin 2000) It is the formation of these mating herds that is of interest, as it has been observed that males will tra vel considerable distances to track females in estrus, similar to the elephant. Although the manatee may mate at any time of the year, peak sexual activity occurs during the warm spring and summer months when manatees have dispersed from their warm water r efuge sites ( Rathbun et al 1995 Reid et al. 1995 ). Therefore, it is assumed that there is a signal given off by the female which alerts males to her receptiveness Although this signal could be visual or auditory, manatee s ha ve limited vision and are reported to be near sighted, having vision only slightly better than river dolphins

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111 (Supin et al. 2001). In addition, manatee vocalization generally occurs in mother and calf communication although it has been observed in mating herds (Hartman 1979). Thei r sound consists of short chirps or squeals in the range of 3 24 kHz (Nowacek et al. 2003) and would most likely be lost in the estuarine environment of the manatee over long distances. A more likely source of this signal would be chemical cues given off b y the female. There have been a number of documented behavioral observations of the manatee suggest ing that femal es produce chemical signals detectable by male s Among the earliest came from Dr. Vosseler, a German veterinary practitioner who was responsibl e for the care of a male and female manatee housed within the same pool together. He noted that when the female was in estrus the male would swim at the bottom of the tank moving his nose and lateral and medial parts of the lip so that the inner lip was ex posed. In addition the male would always find the locations along the area. Hartman (197 9) discussed the prevalence of mouthing between manatees and hypothesized about their chemoreceptive abilities. The body of the behavioral accounts indicates that a female manatee is able to broadcast her reproductive state using secretions and/or excretio ns. The most likely source of this signal is urine, as this is the broadcast used most frequently by mammals (Wyatt 2006). To date there have been no obvious glands detected on the manatee that could provide these secretions outside of the female reproduc tive tract (Don Samuelson Pers Comm, Robert Bonde Pers Comm). The gut transit time of

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112 approximately one week (Larkin et al. 2007) would not be conducive to signal transmission through feces, while it could explain the long time spent in mating herds. Acco rding to current anatomical evidence, the Florida manatee, Trichechus manatus latirostris has taste buds in the foliate papillae of the tongue root (Levin et al. 2002) and a reduced olfactory bulb (Mackay Sim et al. 1985 Reep et al. 2007) but no vomerona sal organ (Mackay Sim et al. 1985). Anecdotally the manatee appears to be able to distinguish between different salt concentrations when locating freshwater and may also use taste in their food choice along with tactile stimuli. In addition it has been hypothesized that the male manatee, has the chemoreceptive ability to track a female in estrus (Reep and Bonde 2006) This project was the first experimental assessment of the use of chemical signaling for reproduction by any marine mammal and specifically the manatee. This study will use male reaction to female urine to elucidate the chemosensory ability of the manatee and the potential location of reception for these signals We expect a behavioral response that may include reproductive or chemosensory beh aviors from the male manatees to female manatee urine. Methods Urine samples came from four captive female manatees that were trained for urine collection (Horikoshi 2004). The reproductive state of the female manatee urine samples used, whether estrus or anestrus, was determined using urinary hormone levels as measured with radioimmunoassays run by the endocrinology laboratory of the Smithsonian National Zoological Park ( Figures 5 1,5 2, and 5 3). Antibodies for estradiol, progesterone luteinizing, and follicle stimulating hormone were used to i ndicate reproductive cyclicity Hormonal analysis determines the phase of estrus for a

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113 particular female at the time of collection and the behavioral tests specify which samples contain a signal of biological inte rest to the male. Figure 5 1. Urinary hormone levels of Lorelei, a female Florida manatee. Luteinizing hormone (LH), Follicle stimulating hormone (FSH), Estrogen (E1S) and Progesterone (PG) were all measured with radioimmunoassays. The red boxes denote estrus peaks and the black box anestrus periods. Figure 5 2. Urinary hormone levels of Charlotte, a female Florida manatee. Luteinizing hormone (LH), Follicle stimulating hormone (FSH), Estrogen (E1S) and Progesterone (PG) were all measured with radioimmunoassays. The red boxes denote estrus peaks and the black box anestrus periods.

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114 Four series of trials were run with a total of five males assessed and three 1). None of the males as sessed had ever been exposed to or mated with a reproductively active female. Each set of trials had a slightly different technique for applying urine to the tank and observing the males due to f acility arrangement. In general, the study used a double blin d, continuous sampling method (Martin and Bateson 2007), recording the start time of each individual behavior as outlined on the ethogram (Table 5 2). This chart was developed from preliminary observations and previous studies examining manatee and elephan t behavior (Bagley et al. 2006, Gomes et al. 2008, Horikoshi Beckett and Schulte 2006, King and Heinen 2004). Figure 5 3. Urinary hormone levels of Sara, a female Florida manatee. Luteinizing hormone (LH), Follicle stimulating hormone (FSH), Estrog en (E1S) and Progesterone (PG) were all measured with radioimmunoassays. The red boxes denote estrus peaks and the black box anestrus periods.

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115 The male manatee(s) was isolated in the medical pool and observed for three minutes for respiratory rate and beh avior without contact Following baseline collection a burette was opened containing 5 10ml of one of the following: salt or deionized water (positive controls in a fresh water or saltwater enclosure, respectively), non estr o us or estr o us urine. The soluti on was released over one minute for the ten milliliter samples and over thirty seconds for the five milliliter samples. Using the ethogram, the observer that was blind to the sample recorded from the opening of the burette both the respiratory rate and the start time for each behavior continuously over five minutes The behavior was videotaped for retrospective analysis and all trials at a given facility were completed within a one week time frame. If an interesting behavior was observed, recording continued for an additional 5 10 minutes. Table 5 1. Male manatees assessed with female manatee urine Snooty was isolated while the other males were observed simultaneously Facility Male(s) Male Current Age/Age at initial captivity Dates trial run Female Urine Estr o us/Anestr o us South Florida Museum Parker Aquarium Snooty 63/Birth August 9th 14th, 2009 Lorelei 5 21 03/7 25 03 August 15th 22nd, 2011 Lorelei 5 21 03/7 22 03 Sara 6 2 05/4 18 05 Mote Marine Laboratory Hugh Buffett 27/Birth 24/Birth November 15th 19th, 2010 Lorelei 7 11 03/7 2 03 Lorelei 5 3 03/4 5 03 Lorelei 6 22 03/7 18 03 Disney Epcot's the Living Seas Lou Vail ~20/~a year ~20/~a year July 22nd 26th, 2011 Charlotte 4 10 05/10 13 05 Sara 4 4 05/9 18 05

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116 Table 5 2. Ethogram of behavior categories performed by captive male West Indian manatees exposed to urine of female manatees. Developed from Bagley et al 2006. Behavior categories specific behaviors Definition Active Circular movement (CM) Moves around enclosure in a circular motion Circular Pectoral (CP) Moves around enclosure in a circular motion using pectoral flippers. Interaction at Gate (GI) Nose to gate with or without animals on the other side. Lift (LI) Removes upper body from tank using pectoral flippers Bottom Swim (BS) Swimming along bottom of enclosure Continuous Roll (CR) Swimming and rolling continuously Emerge (EM) Lifts head out of water Gate Interaction (GI) Swimming at gate separating medical pool and main enclosure Chemosensory Search Opens and moves lips to allow water passage at bottom of enclosure Roll (RO) Turns body completely within water column only chemosensory to Snooty Sniff (SN) Breaths in through nostrils rapidly Continuous Roll Corner(C Roll C) Rolls continually in corner at which sample applied Search and C o prophagia (Search/Cap) Searching behavior followed immediately by ingestion of fecal Inactive Surface Rest (SR) No movement at water surface Bottom Rest (BR) No movement at tank bottom Nose to Wall (NW) Presses nose to wall Submerged (SU) Submerges below the surface after activity Column Rest (Col R) No movement within water column Surface Rest at Gate (SR GI) No movement at gate separating medical and main pool Socialize Socialize Both (SO Both) Both Lou and Vail Participate in Socialization Socialize Lou (SO Lou) Lou attempting to socialize with no reciprocation from Vail Socialize Vail (SO Vail) Vaile attempting to socialize with no reciprocation from Lou Other Other (OT) Displays a behavior not defined by the ethogram

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117 The trials occurred at the same time of day to decrease time of day effects. There was a 25 45 minute break between each of the three assays within a given day depending on the size of the tank and constraints on animal availability. The protocol of three trials (positive control, estrous and anestrous urine) presented once a day was repeated over the course of five days. Only following complete data analysis that included verification of behaviors on video was the researcher informed about the identity of the samples added for an individual assay. Variation from the general protocol included the first series of data collection with Snooty in August 2009 were collected using both a positive (salt water) and negative (tank water) control. Behavior was observ ed for fifteen minutes instead of three prior to adding sample to the water and observed for twenty minutes instead of five after the sample was added to the water. Only two trials of the four (negative control, positive control, anestr o us and estr o us urin e) were run during a day with a separation of at least one hour to allow tank filtration. E ach of the four trials was repeated three times over the course of six days. Hugh and Buffett could not be isolated in the medical pool and so were called back to t he pool at the beginning of both pre and post sample addition. Also during a few of the trials pipettes were used for sample addition instead of burettes: November 15 th 2010 observing Hugh and Buffett, July 23 rd and 24 th observing Lou and Vail, and August 16 th and 17 th observing Snooty. Results Positive responses were observed from t wo male manatees, Snooty ( Figure 5 4) and Lou ( Figure 5 5), to female manatee estr o us urine and little to no reaction to anestr o us urine. Reaction to estr o us urine included more time spent below the surface

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118 exploring the bottom of the tank in a behavior termed searching. In the case of Snooty the behavior included rolling, which was not observed in any other trials during the August 2009 series of trials. Specifically, Snooty res ponded to the addition of estr o us urine during one of the three trials in which it was added. Snooty spent 359 seconds of the total 900 seconds searching and rolling. This in comparison to the salt water sample reaction in which he spent 150 seconds search ing and did not roll at all. Lou responded to the addition of estr o us urine during four out of the five trials in were calculated as a percent of time spent on a behavior. During the first estr o us sample he spent 47% of his time searching and rolling beneath the drip. On the second day, following estr o us urine addition Lou spent 34% of his time searching. On the third day he spent 24% of his time searching and on the fourth day 39% of his time searching and rolling beneath th e drip. The reaction on the final day of trials was not considered a response because Lou had exhibited searching behavior accounting for 27% of his time during the previous water trial therefore; the 8% of time spent searching following estr o us sample add ition was not considered a genuine reaction. Neutral responses to the estr o us urine were observed d uring the Snooty August 2011 trials ( Figure 5 6). The male manatee Vail, who is housed with Lou and was assessed simultaneously as him, also had a neutral re sponse to the urine. He spent a majority of his time swimming in large circles and rolling or interacting with Lou. He also exhibited co prophagia which accounts for most of the chemosensory response outlined in Figure 5 7. Neither Hugh nor Buffet exhibite d a response to the urine ( Figure 5 8). Three out of the five days of trials were spent begging for food or attention.

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119 Figure 5 4. Behavior of Snooty in 2009 in response to female urine from different reproductive time points. A) Summary o f time spent on activities deemed Interaction (GI), Lift (LI), Emerge (EM), Swim and Movement (MVMT). Rest includes: Stop, Submerged (SU), Surface Rest (SR), Bottom Rest (BR), and Nose to Wall (NW). Chemosensory includes: Roll (RO), Search, and Sniff (SN) A B

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120 Figure 5 5. Behavior of Lou in 2011 in response to female urine from different reproductive time points. A) Summary of time spent on activities deemed ing (PS), Bottom Swimming (BS), Rise, Continuous Roll (C.Roll), Roll, and Gate Interaction (GI). Rest includes: Surface Rest at Gate (SR GI), Bottom Rest (BR), Surface Rest (SR), and Column Rest (Col. R). Chemosensory includes: Search, and Continuous Roll Corner (C.Roll C). Socialize includes: Socialization for both (SO Both), Socialization from Lou (SO Lou) and Socialization from Vail (SO Vail). B A

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121 Figure 5 6. Behavior of Snooty in 2011 in response to female urine from different reproductive tim e points. A) Summary of time spent on activities deemed B) Exact time spent on all observed Clockwise (CP CW ), Circular Pectoral Swimming Counter Clockwise (CP CC W), Gate Interaction (GI), Circular Pectoral Aggressive Swimming (CP Ag), Lift (LI), Swim and Bouncing Aggressive (B/AG ) Circular Movement (CM) Rest includes: Stop, Emerge (EM), Submerged (SU), Surface Rest (SR), Bottom Rest (BR), and Nose to Wall (NW). Chemosensory includes: Roll (RO), Search A B

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122 Figure 5 7. Behavior of Vail in 2011 in response to female urine from diff erent reproductive time points. A) Summary of time spent on activities deemed time spent on all observed (BS), Rise, Continuous Roll (C.Roll), Roll, and Gate Interaction (GI). Rest includes: Surface Rest at Gate (SR GI), Bottom Rest (BR), Surface Rest (SR), and Co lumn Rest (Col. R). Chemosensory includes: Search and Search followed by co prophagia (Search/Cap) Socialize includes: Socialization for both (SO Both), Socialization from Lou (SO Lou) and Socialization from Vail (SO Vail). A B

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123 Figure 5 8. Behavior of male manatees to female urine from different reproductive time points in 2010. A) Hugh and B ) Buffett B A

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124 Discussion Snooty is the oldest known manatee at 63 years old and has been in captivity his entire life. He has never mated or been in captiv ity with a reproductively active female: h owever, he is housed occasionally with female calves that are being rehabilitated before release. During the 2009 trial in which there was a heightened reaction he was housed with a female that was approaching her release date and was nearly the size of a juvenile. He was also exhibiting behaviors outside of the study period that were reproductive in nature. However, during the 2011 trials he was not exh ibiting these behaviors and was not house d with a larger female calf. This may explain his response in 2009 but not 2011. Regardless, his response indicates that there is an innate chemosensory trigger for male manatees that can be found in female manatee estr o us urine. L ou has been in captivity since he was a calf and also has never mated with or been in the same enclosure as a reproductive ly active female. His behavior corroborates the results of Snooty and his younger age may explain the more consistent reactions to the estr o us urine. Also, due to time constraints on the availability of Lou and Vail there were only 25 30 minute breaks between trials. This may explain the continued chemosensory behavior of Lou in assays following estr o us urine input. Vai l also in captivity since he was a calf and never mated, exhibited chemo sensory behavior that was predominantly co prophagic, which is common in his consumed validate s the use of the term search to locate a chemical of interest. The lack of response from Vail to the estr o us urine is likely due to his submissive nature within

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125 the hierarchy of the housing. Lou is the dominant male and this is an indication of why Vail di d not react. Hugh and Buffett both born in captivity and never mated, are trained to react to stimuli and be rewarded. They spent a majority of the trial time begging for food or attention from the researchers and viewing public. They also could not be is olated in the medical pool which made observations difficult because the entire main enclosure could not be observed from a single vantage point This is why only their location and general behavior are reported. All of these males have been in captivity their entire adult lives and have never mated or been exposed to a reproductive female. Therefore, these results are preliminary and should be repeated with wild males or those that are in captivity but are known to have mated or been in the wild as reprod uctive adults. Despite the naivet of the males, it is apparent that male manatees are capable of detecting females in estrus. The fact that Snooty at 61 reacted to estr o us urine after having never mated indicates how strong this instinct is and it is lik ely that if this is repeated with reproductively active males the results would be more conclusive. The behavioral reaction occurs entirely underwater, indicating that the chemicals are brought to receptors through aqueous transmission. This could be done through retronasal delivery from the oral cavity through the caudal portion of the nasal tract to the olfactory epithelium or the taste buds could be involved in the sensing of the chemical(s) of interest within the urine. There also could be a mechanism not yet understood. Researchers have previously alluded to the idea that marine mammals use chemoreceptors that may vary from terrestrial mammals (Oelschlager 2008b, Friedl et

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126 al. 1990, Kuznetsov 1990). The manatee presents an opportunity to study this fur ther and determine if their taste buds, olfactory epithelium, or other chemoreceptors are responsible for this behavioral response. physiology a further understanding of the manatee This will aid in determination of environmental factors that chemoreceptive abilities (Sandahl et al. 200 7). In addition, with an increased understanding of the physiology of manatee chemoreception and its manifestation as behavioral changes a more extensive understanding of its role in wild manatee reproduction is possible.

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127 CHAPTER 6 CONCLUSIONS Experimentation on an endangered marine mammal is difficult for many and logistical reasons. Despite this the Florida manatee has provided an opportunity to document for the first time chemoreceptive systems and behavioral responses of a fully aquatic mamm al in relation to chemical communication. These results will help to direct future reproductive studies of the manatee and other marine mammals. The male Florida manatee has demonstrated behaviorally that it responds to chemical cues in the urine of femal es. The chemical analysis of this urine has demonstrated a significant difference in 64 atomic weights of chemicals present in estr o us compared to anestr o us urine (Appendix ) Through further studies these masses will be analyzed to determine the most likely metabolite candidates for causing the behavioral responses recorded in this study. Then those chemicals can be isolated for more critical analysis techniques including the use of tandem mass spectrometry to id entify a more complete representation of the composition of those chemicals. This is the first step to identifying a chemical compound that is expressed and perceived by a fully aquatic marine mammal. The expression of these chemicals has been demonstrate d in urine but is likely also found in the anal glands. The suggestion of a seasonal change in gland size and productivity indicates that manatees may be using their glands to communicate with mates. This is similar to the behavior observed in the river ot ter Lutra lutra (Gorman et al. 1978). However, the otter deposits its secretions on land, while the manatee is depositing into an aqueous environment therefore the chemicals are likely very different

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128 in composition This is the first d escription of these glands in a fully aquatic mammal but they are likely found in many other marine mammal species. The mode of reception for these chemical signals is unknown. Their behavioral reaction appears to be entirely underwater, indicating that the chemicals are bro ught to receptors through aqueous transmission. This could be through retronasal delivery to the olfactory epithelium or the taste buds could be involved in the sensing of the chemical(s) of interest within the urine ( Figure 6 1) There also could be a mec hanism not yet understood. Researchers have previously alluded to the idea that marine mammals use chemoreceptors that may vary from terrestrial mammals (Friedl et al. 1990, Kuznetsov 1990 Oelschlager 2008b ). These have included allusions to potential sma ll, non taste bud receptors on the tongue (Oelschlager 2008b) or receptors found external to the oral cavity (Kuznetsov 1990). As demonstrated, the manatee has ample taste buds to perceive a chemical signal but the transmission of pheromonal cues has nev er been shown to take place through taste. However, the transmission of the chemicals retronasally to the olfactory epithelium would be difficult because of the valve at the base of the nasal canal. The manatee would have to have excellent control of this valve to allow aqueous solutions to pass from the oropharynx and through the nasopharynx to the olfactory epithelium at a minimum volume of water The manatee does have olfactory epithelium and has demonstrated the ability to sniff at odorants of interest above the water but the manatee does not open their n ares underwater and so direct in put through the nares is unlikely.

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129 Figure 6 1. Overall chemosensory receptor organs within the head of the manatee. Note the foliate papillae and location of taste buds within the soft palate as well as, the apparent taste buds at the orthonasal valve. Arrows represent potential airflow for olfactory detection, both through the nares (1) and retronasal delivery (2). The undescribed mode of chemical receptio n could be related to the solitary chemosensory cell (SCC). These cells are used by terrestrial mammals in the nasal canal and trachea to perceive and respond to irritants including bacteria (Lin et al. 2008, Tizzano et al. 2010 Tizzano et al. 2011 ). They are found within the skin along the entire length of the body in many fish species and are used by fish to detect signals given off by conspecifics (Kortschal 1995). As the manatee is an aquatic mammal it is possible rep resented by terrestrial mammals and fish. manatee. 1 2

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130 It appears that the manatee uses some type of sex pheromone but it is also possible that they have an aggregation pher omone as well. Mating herds are composed of anywhere from a few to 12 or more male manatees pursuing a single female (Hartman 1979, Reid et al. 1995). Males are likely attracted by sound to these herds and by the signal released by the female during estrus ; however other males may also be releasing a signal that over time has evolved into an aggregation pheromone used as and eavesdropping signal Manatees likely do not use territorial pheromones, alarm pheromones, or pheromones for social organization. Wit hin mammals, specific effects that are observed in relation to reproduction include the Bruce, Whitten, Vandenbergh, Lee Boot, and Coolidge effects. Any of these effects that require a high density of animals such as the Lee Boot, which inhibits estrus in females by other females (Vandenbergh 1999) are likely not occurring in wild manatee populations. The Bruce effect is a pregnancy block that results when females s mell urine released by males that did not fertilize the egg (Bruce 1960). This would be coun terproductive in a mating herd because so many un successful males are present. The Whitten effect results in the induction of estrus in adult females by male pheromones while the Vandenbergh effect accelerates puberty in young females through the same male pheromone signals (Ma et al. 1999, Novotny et al. 1999). These effects could o ccur in manatees but would be difficult to assess. Finally the Coolidge mate to increase potential variability in genes ( Johnston and Rasmussen 1984). Female manatees are generally pursued during mating herds and will become evasive during

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131 them ( Hartman 1979, Reid et al. 1995 ). This behavior does not indicate an interest in a male as would be expected during the Coolidge effect. In general, the manatee has a reduced sense of smell but an apparent ly heightened sense of taste with in a localized region the tongue root, of the oral cavity. An aquatic environment is more conducive to those chemicals that are perceived by the taste buds The reduction of olfactory use is evident both anatomic ally with the loss of olfaction in odontocetes (Oeschlager and Oeschlager 2008b), and genetic ally with reduced olfactory receptors in fish 50 100 (Ngai et al. 1993, Barth et al. 1996) versus terrestrial mammals 400 1000 (Parmentier et al 1 992, Ressler et al. 1994). There also may be a correlation between the type of food an animal eats and their degree of taste. For example, the herbivorous bovine tongue has many more taste buds (14,765 21,691) than the omnivo rous rhesus monkey (8000 10000) : the manatee ( 3,233 31,140) may have a similar amount of taste buds to the bovine. However, future studies should calculate density of taste buds within an area to take into account the size of the tongue, or area in which taste buds are located. The mana tee likely has the highest concentration of taste buds within a small area than any other species yet examined. From what has been observed during the behavioral trials it appears that a manatee can sense a relatively small amount of urine, five ml, from up to three meters away. In a natural situation it is likely that manatees can sense the much larger normal urine bouts from distances larger than three meters. They are probably using chemical sensing for distances less than a hundred meters but this is likely the primary initial sense that males use to locate females during traveling.

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132 Locating females for copulati on is likely a combination of learned migratory patterns, to areas where males have been successful in the past, in addition to encountering acoustic cues from other males and perhaps the receptive female, as well as chemical signatures of both the recepti ve male and other females. Vision and touch are likely involved in the act o f mating but not locating mates or mating herds. From the current research it is unclear which chemoreceptors: olfactory, taste, both or something nsible for the behavioral response. Anecdotal evidence in addition to the life history patterns of marine mammals indicates that they are capable of chemoreception for both feeding and reproduction Taste is most likely used by all marine mammal species fo r detecting salt water gradients and supplementing their food preference. It may also be used in detecting signals given off by other animals either for detection of prey or mates. Many marine mammal species would be served through the use of taste, solita ry chemoreceptors such as those found on fish or some other chemical detection mechanism to aid in the detection of mates and the assessment of food. More comprehensive and comparative research is needed in all genera of marine mammals for both taste and s mell but the area that is least researched and has been most hypothesized in these animals is the use of chemoreception for chemical signaling in reproduction. The Florida manatee demonstrates that this is a possibility and further research is needed. Fin ally, more research is needed in general on the mechanisms of chemoreception. Comparatively little is known about these complex mechanisms compared to the other senses of sight, audition and touch. It would be beneficial to determine what marine mammals ar e capable of sensing compared to their terrestrial

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133 and aquatic counterparts. Aquatic mammals are an important evolutionary link and would provide information about the development and loss of chemoreception that would aid in overall studies of these senses The aquatic environment is an ideal medium for the use of chemosensory systems and the use of these by marine mammals correlates with what has long been hypothesized.

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134 APPENDIX CHEMICAL ANALYSIS OF FEMALE FLORIDA MANATEE TRICHECHUS MANATUS LATIROSTRIS URINE The first step in identification of pheromones released by the manatee for chemical signaling is analysis of the urine. Similar experiments have included analysis of goldfish pheromones in which six females were utilized to verify the release of th e dihydroxy 4 pregnen 3 one (Dulka et al. 1987) In an aquatic newt a sample size of fifty was used to identify the peptide pheromone, s odefrin, released by the male red bellied newt (Kikuyama et al. 1995). The smaller sample size of the goldfish was reliant on previous research while the larger sample size of the newt was to collect enough secretion to allow for beh avioral assays and analysis. The current study on manatees will require a sample size in between these numbers most li kely close to the nine individuals required to determine the identity of the elephant pheromo ne (Z) 7 d odecen 1 yl a cetate (Rasmussen et al. 1996). To begin the identification of potential chemicals in female manatee urine that may be involved in attracti ng a male for reproduction a metabolomics approach was certain conditions, for example disease or a natural state such as pregnancy. The metabolome or collection of metabo lites can represent an individual as a whole or certain tissues, systems or cells within that organism. It represents both the genomes effect on the system and the natural or external conditions (Rochfort 2005). Through comparison of variation in the metab olome of an organism in differing natural states such as those related to reproduction one can develop an understanding of the changes taking place and potential chemical cues used by conspecifics.

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135 A global urine metabolomics approach was used to compare me tabolome variation between estr o us and anestr o us female manatee urine. Urine samples came from four captive female manatees that were trained for urine collection (Table A 1) (Horikoshi 2004) The anestr o us and estr o us state were determined using urinary hormone levels as measured with radioimmunoassays. Also, three estr o us samples; Lorelei 5/21/03, Charlotte 4 10 05 and Sara 4 4 05 were confirmed to provoke a behavioral response in male manatees. The chemist running the analysis was not aware of which group of samples were estr o us and which were anestr o us. Table A 1. Manatee urine samples used for chemical analysis. Anestr o us Estr o us Lorelei 7/25/03 Lorel e i 5/21/03 Lorel e i 6/22/03 Lorel e i 6/30/03 Lorel e i 5/3/03 Lorel e i 7/4/03 Lorel e i 7/11/03 Lorel e i 5/17/03 Charlotte 10/13/05 Charlotte 5/17/05 Charlotte 5/30/05 Charlotte 4/10/05 Sara 5/22/05 Sara 5/7/05 Sara 4/18/05 Sara 6/2/05 Sara 9/18/05 Sara 9/24/05 Sara 9/13/05 Sara 4/4/05 Holly 8/3/10 Holly 2/3/10 One concern with the urine samples was the 6 8 years storage time and 2 3 known freeze thaw events of the current samples. However, t here have been numerous studies targeting the effects of storage and freeze thaw on metabolite profiles which indicate tha t for the purposes of qualitative evaluation, there is minimal effect In one such experiment the e ffect of up to nine freeze thaw events, storage in a 20 o C versus a 80 o C freezer and storage for up to six months had no effect on the stability of the urin e while significant differences were observed following 48 hours or more of the sample at room temperature (Gika et al. 2008). The changes observed in the urine were

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136 quantitative with increases in some and decreases in other well known metabolites. Therefo re, it is likely that with proper storage techniques the impact of bacteria and any freeze thawing would not affect the results of a qualitative study such as this. Urine was analyzed using a reverse phase high performance liquid chromatography (HPLC) coup led to an Agilent time of flight (TOF) mass spectrometer with a positive electrospray ionization The samples were run in triplicate. This combination allows for the separation and measurement of molecular weight of metabolite ions within a sample. The mas s spectrometer has a mass accuracy of less than two ppm which will enable metabolic database searching for identification of small molecule metabolites and, therefore, will work well with pheromones which have a small molecular weight typically between 80 and 300 (Wyatt 2006). Following mass measurements, t he amount of information generated require d multivariate statistical tests to indicate masses of interest. Chemometric analyses including a principal components analysis (PCA) and partial least squares r egression were employed and are the most commonly used statistical techniques for metabolomics data. Both of which allow for analysis and modeling of complex data sets that is, datasets that include multiple points of interest such as species, time of coll ection, control versus experimental group etc. These techniques also account for noisy, incomplete and collinear data structure analysis. PCA allows for comparison of datasets in a visual model plane that indicate groupings, trends or outliers among the d ata. Partial least square regression is similar to PCA but instead of providing a number of different dimensional variables it expresses the predicted and observed va riables as a linear regression (Trygg et al. 2007).

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137 The peaks, which represent the time t hat an ion traveled and therefore its molecular weight were aligned and compared using an unpaired t test. Out of 1,163 aligned peaks, 64 were considered significant and run in a PCA and partial least squares discriminant analysis (PLS DA). The PCA showed nice separation between groups A anestr o us and B estr o us ( Figure A 1) urine as did the PLS DA ( Figure A 2). Figure A 1. A PCA plot demonstrating separation between group A anestr o us urine on the left and group B estr o us urine on the right. Figure A 2. A PLS DA analysis demonstrating separation in group A anestr o us urine on top and group B estr o us urine on bottom.

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138 We will next focus on masses that are significantly different and found in the estr o us urine samples that evoked a behavioral response. Then, to determine which metabolite a mass may represent we will search for it using one of several available databases There are thousands of potential metabolites expressed in urine and these vary between species, individuals and according to environmental and body conditions. Databases listing known metabolites and their chemical properties are the best source of identification currently available. The human has the most studied metabolome with several databases listing metabolites. The human metabolome database (HMDB) has over 2100 human metabolites from urine, blood, and cerebrospinal fluid listed including over 90 descriptive parameters for each metabolite (Wishart et al. 2007). The consortium for metabonomic toxicology, COMET, database is also human specific and includes metabolites representing drug toxicity (Rochfort 2005). Maintained by the National Magnetic Resonance Facility in Madison, Wisconsin the Madison Qingdao Metabolomics Consortium Database (MMCD) pulls information from public databases such as Kyoto Encyclopedia of Genes and Genomes (KEGG) and PubChem to provide over 50 parameters on metabolites from a variety of species (Cui et al. 2008). There is also a bovine database being developed that will provide the most comparable me tabolites for manatees. We would also like to expand our criteria to include more potential chemical masses. Those shown above were present in a least half the samples, but there was already a natural division of the samples in half, it would be more accu rate to put the constraint of presence in at least a quarter of the samples. This would mean that it would be found in at least half of the estr o us or half of the anestr o us samples. Following

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139 identification of masses of interest those ions at the mass and retention time will be isolated and analyzed using tandem mass spectrometry. This will give a more accurate description of the components of the chemical. The separation between the two groups A anestr o us and B estr o u s is encouraging and indicates that there are metabolites that serve the purpose of signal expression. To identify these metabolites we need a larger sample size closer to the six or nine individuals used in goldfish (Dulka et al. 1987) and elephant (Rasmussen et al. 1996) analysis We also need more examples of estr o us and anestr o us urine, e g ., various dates to increase the samples available for analysis.

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156 BIOGRAPHICAL SKETCH Meghan Lee Bills was born in Oswego, New York in 198 2 to parents Deborah (Koester) Bills and Richard Lee Bills of Fair Haven, NY Meghan was raised in Fair Haven, NY and graduated from Red C reek Junior/Senior High School in 2001. She attended college at the University of Delaware during which time she completed a senior thesis analyzing spontaneous ovarian contractions in several fish species under the supervision of Dr. Malcolm Taylor Megha n graduated with an Honors Bachelor of Science degree with distinction in 2005. She went on to receive a Master of Science degree in marine b iology from Nova Southeastern University in 2007 During her masters she focused on bottlenose dolphin distribution along the Southeast coast of the United States under the supervision of Dr. Edward Keith She then received her PhD in 2011 from the University of Florida Aquatic Animal Health Program of the College of Veterinary Medicine under the supervision of Dr. Iskande Larkin and Dr. Don Samuelson.