MORPHOMETRIC VARIATION IN THE PHARYNGEAL TEETH OF ZEBRAFISH (Panic rerio Cyprinidae) IN RESPONSE TO VARYING DIETS by JASON MICHAEL MILLER, B.S. A THESIS IN ZOOLOGY Submitted to the Graduate Faculty of Texas Tech University in Partial FulfiUment of the Requirements for the Degree of MASTER OF SCIENCE Approved December, 1999 ©1999 by Jason Michael Miller All rights reserved ACKNOWLEDGEMENTS This research would not have been possible whhout the encouragement and support of several mdividuals. I would like to thank Dr. Richard Strauss for introducmg me into the world of biological research, as weU as contributing endless hours with me on aU aspects of this research. I am extremely grateful for having the opportunity to work and learn in his laboratory. I would also like to thank my other commhtee members. Dr. Marilyn Houck and Dr. Gene Wilde, for theh msight and patience throughout the duration of this research, always making h inteUectuaUy challenging. I would like thank Texas Tech Univershy Department of Biological Sciences in making available to me seemingly mexhaustible resources throughout my graduate career. To lab partners and good friends: Ozlen Konu, Maddalena Can^ioni-Noack, Joao Ohvera, Christian Schroder, David Kippie, Kelly Robinson and Kelly Johnson: each of you were sources of insphation and support throughout this study, helping me remain calm during those frequent times of stress. I am mdebted to Mark Grimson for his help in using the SEM, and Josh Pritchard for his work in getting the SEM negatives scanned into the computer. I would also like to thank Dr. Joseph Kuban for providing me with my fu"st introduction into the world of biology and instilling in me the intellectual curiosity necessary to pursue it this fer. A special debt of gratitude is owed to my entire femily, especially to my mother, father, and sister. I will always be thankful to each of you for your love, encouragement, and confidence that have given me the opportunity to pursue and achieve this goal. I would finally like to thank Amy, whose support and patience were beyond anything I could ever ask: her love and understanding during the duration of this research, which kept us apart, was a tremendous source of strength and something for which I will be forever gratefiil. 11 TABLE OF CONTENTS ACKNOWLEDGEMENTS u LIST OF TABLES vi LIST OF FIGURES vii CHAPTER I. INTRODUCTION 1 The Pharyngeal Denthion of Fish 1 The Zebrafish: A Historical Perspective of the genus Danio 2 The Zebrafish: A Model Organism 3 Phenotypic Plasticity 4 Research Objectives 4 n. MATERL^LS AND METHODS 6 Experhnental Design 6 Breeding Protocol 6 Collection and Care of Embryos 7 Care and Rearing of Zebrafish Fry 8 Selected Diets and Feedmg Procedures 9 Sanq)lmg Protocol 10 Modifications m 2^brafish Collection 10 Histological Preparation 11 Imaging Protocol for Skeletal Measurements 12 Dighizing and Measuring of Skeletal Structures 12 Dissection of Pharyngeal Arch and the Preparation of Teeth for SEM 13 SEM Use and Photographing the Arches 14 Digitizing of the Pharyngeal Teeth 14 Methods of Analysis 15 m III. RESULTS 24 Analysis of Skeletal Body Measurements 24 Principal Component Analysis of Skeletal Measurements 24 Analysis of Body Size for Varying Diets 26 Physical Description of the Pharyngeal Teeth m the Zebrafish 26 Location and Poshion of Teeth m the Oral Cavity 26 Physical Appearance of the Teeth 27 Analysis of Tooth Data 27 Principal Component Analysis for the pooled Sample of Teeth 27 Analysis of Tooth Data usmg Diet as an Envhonmental Factor 31 Tooth 1 32 Tooth 2 33 Tooth 3 33 Tooth 4 33 Tooth 5 34 Tooth 6 34 Teeth 7 and 8 34 Tooth 9 35 Teeth 10 and 11 35 Predicted Tooth Triangles 35 IV. DISCUSSION AND CONCLUSION 78 Overview of Variation Caused by the Three Treatment Diets 78 Diet # 1 Artemia (Brine Shrimp) 78 Diet # 2 Egg Yolk 79 Diet # 3 Tetra Mm® Flake Food 80 Correlations between the Studies of Cichlids and Zebrafish 81 Conclusion 82 IV LITERATURE CITED 84 APPENDIX 87 LIST OF TABLES 2.1 A general representation of important nutritional characteristics from each of the three selected diets. *(The egg yolk is based on an average sizeofl6.6g) 16 2.2 Individual landmarks that were dighized on both the dorsal and lateral skeletal images of the zebrafish. 17 2.3 Characters measured by the dighized landmarks for both dorsal and lateral views. 18 2.4 Tooth characteristics measured by the plotted landmarks for each pharyngeal tooth. 19 3.1 Analysis of covariance (ANCOVA) resuhs for the measure of body size modeled on diet, using age as a covariate. 37 3.2 Proportion of variation that can be accounted for by the principal con:qx)nents of the body measurements. 37 3.3 Loadings, with 95% confidence mtervals, for each of the body principal components. 38 3.4 Body allometry scores for the thirteen measiu^ed body characteristics. 39 3.5 Proportion of variation that can be accounted for by the principal components for the pooled sample of teeth. 44 3.6 Loadings with theh confidence mtervals, set at 95%, for each of the principal conqwnents of the pooled tooth data. 44 3.7 Tooth allometry scores for the seven-measured tooth characteristics. 45 3.8 Proportion of variation that can be accounted for by the principal components for the pooled tooth data within an uidividuaL 51 3.9 Loadings with thek confidence intervals, set at 95%, for each of the pruicipal components of the pooled tooth data wdthm an mdividual. 51 3.10 Tooth allometry scores for the seven measured tooth characteristics centered within mdividuals. 52 3.11 MANOVA resuhs for differences m teeth due to diet. 5 8 VI LIST OF HGURES 2.1 Data plot showing the twelve individual san^le dates for each diet in all three breeding units for a total sample size of 108. 16 2.2 Lateral image of cleared and stained zebrafish wdth dighized points and character line measures shown. 20 2.3 Dorsal image of cleared and stained zete^fish with dighized points and character line measures shown. 20 2.4 Border outline landmark distances around a pharyngeal tooth which correspond to the selected measured characters given in Table 2.4 21 2.5 Straight-line landmark distances around a pharyngeal tooth which correspond to the selected measured characters given in Table 2.4. 21 2.6 SEM photograph right pharyngeal arch (lateral view). Tooth numbers 1-5 shown left to right. 22 2.7 SEM photograph left pharyngeal arch (lateral view). Tooth numbers 1-5 shown right to left. 22 2.8 SEM photograph right pharyngeal arch showing all three rowrs of teeth- numbering is from right to left starting with back row and working forward. 23 2.9 SEM photograph left pharyngeal arch showing all three rows of teeth, numbering is from left to right starting with back row and working forward. 23 3.1 Scatter plot of principal components 1 and 2 for general body size. 40 3.2 Vector plot showing the relationships of principal con:q>onents 1 and 2 of the body measurements for both dorsal and lateral views. 40 3.3 Scatter plot of principal con^nents 2 and 3 for general body size. 41 3.4 Vector Plot showing the relationships of principal con^nents 2 and 3 of the body measurements for both dorsal and lateral views. 41 3.5 Histogram representmg the frequency of body size with respect to principal continent 1. Arrow mdicates the mean of the data. 42 3.6 Regression plots of age versus general body size for each of the three diets: brine shrimp (b), flake food (f), and egg yolk (e). 42 3.7 Allometric coefficicents and their confidence intervals for the measured dorsal and lateral body characteristics. 43 3.8 Scatter plot of all teeth on principal con^nents 1 and 2 for pooled tooth data. 46 Vll 3.9 Convex hulls encompassing the variation of each tooth due to principal components 1 and 2. Numbers and centroid points are shown for each tooth. 46 3.10 Plot of convex hull centroids for the eleven teeth for the pooled tooth sample based on principal conqx)nents 1 and 2. 47 3.11 Vector Plot for the seven measured tooth characteristics for the pooled sample m relation to principal components 1 and 2. 47 3.12 Scatter plot of aU teeth on principal components 2 and 3 for the pooled tooth data. 48 3.13 Convex hulls encompassing the variation of each tooth due to principal con^nents 2 and 3. Numbers and centroid points are shown for each tooth. 48 3.14 Plot of convex hull centroids for the eleven teeth based on principal components 2 and 3. 49 3.15 Vector plot for the seven measured tooth characteristics in relation to principal components 2 and 3. 49 3.16 Allometric coefficicents and theh surroimding confidence intervals for the measured tooth characteristics, (jraph represents analysis across all individuals. 50 3.17 Scatter plot of pooled sample of teeth within individuals and the variation due to principal components 1 and 2. 53 3.18 Convex hulls encon^assing the variation of each tooth due to principal components 1 and 2. Numbers and centroid points are shown for each tooth. 53 3.19 Plot of convex hull centroids for the eleven teeth, centered within individuals, based on principal conqx)nents 1 and 2. 54 3.20 Vector pk)t for the seven measured tooth characteristics, centered within individuals, in relation to principal components 1 and 2.