MORPHOLOGY OF THE OLFACTORY APPARATUS IN LEPTOCEPHALUS LARVAE by MOLLY ANN WIGHTMAN B.S., Florida Institute of Technology A thesis submitted to the Department of Biological Sciences of Florida Institute of Technology in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in BIOLOGICAL SCIENCE Melbourne, Florida July 2018 MORPHOLOGY OF THE OLFACTORY APPARATUS IN LEPTOCEPHALUS LARVAE A THESIS By MOLLY ANN WIGHTMAN Approved as to style and content by: Jonathan Shenker, Ph.D., Chairperson Ralph Turingan, Ph.D., Member Associate Professor Professor Department of Biological Sciences Department of Biological Sciences Kelli Hunsucker, Ph.D., Member Richard Aronson, Ph.D. Research Assistant Professor Professor and Head Department of Ocean Engineering Department of Biological Science and Sciences July 2018 ABSTRACT MORPHOLOGY OF THE OLFACTORY APPARATUS IN LEPTOCEPHALUS LARVAE By Molly Ann Wightman, B.S., Florida Institute of Technology Chairperson of Advisory Committee: Jonathan Shenker, Ph.D. Leptocephalus larvae are a unique larval form that unites the Elopomorph fishes. This larval form has morphological and cellular characteristics that differ from most other teleost fishes. The visual system of leptocephali rod-dominated retinas, leading to poor photopic vision and low visual acuity that would hinder feeding on planktonic prey. Recent studies indicate they feed marine snow, which is composed of mucilaginous materials, bacteria and plankton. Their low visual acuity raises the question of how these organisms find this gelatinous food source in the wild. I hypothesize that these larval fishes use chemoreception in order to find marine snow, and that their olfactory apparatus thus differs morphologically from other teleost fishes. A variety of settlement-stage pelagic larvae were collected from Sebastian Inlet State Park in Florida and Andros Island, Bahamas. Leptocephalus larvae were represented by tarpon (Megalops atlanticus), ladyfish (Elops saurus), bonefish iii (Albula vulpes), and speckled worm eels (Myrophis punctatus). Three species with non-leptocephalus larvae were examined: Atlantic menhaden (Brevoortia tyrannus), Atlantic croaker (Micropogonias undulatus), and pinfish (Lagodon rhomboides). Their external olfactory and visual systems were analyzed using Scanning Electron Microscopy. Morphometric data were collected and compared to make comparisons between larval types regarding the dimensions and structures of these sensory organs. Developmental differences were also observed by comparing SEM images of larval and juvenile fishes. Three out of the four species of leptocephalus larvae had large, exposed olfactory pits, while speckled worm eels and all non-leptocephalus larvae had their olfactory apparatus embedded under the skin of the head, with distinct anterior and posterior nostrils. Although there was no consistent difference among the groups in dimensions of the olfactory apparatus, eye size, or ratio between the sensory organs, the open olfactory pits of tarpon, ladyfish and bonefish larvae would directly expose sensory cells to chemical signals from marine snow. The enclosed olfactory apparatus of the non-leptocephalus species develops during embryogenesis, suggesting that exposed olfactory surfaces are not as important as vision in detecting motile plankton prey. Speckled worm eel larvae entering the estuary had already developed closed olfactory pits and nostrils, presumably in preparation for their rapid assumption of a life style where they bury in sediments during the day. Bonefish and ladyfish showed a similar development of closed olfactory pits and nostrils after metamorphosis within the estuarine habitat. iv Studies of receptor densities, olfactory organ ontogeny, and testing the olfactory sensitivity of live larvae could help to further understand the life history of the Elopomorph fishes, and help with continued conservation and aquaculture efforts. v TABLE OF CONTENTS Page ABSTRACT . iii TABLE OF CONTENTS . vi LIST OF FIGURES . viii LIST OF TABLES . xi ACKNOWLEDGEMENTS . xii DEDICATION . xiii INTRODUCTION . 1 LEPTOCEPHALUS LARVAE . 4 CHEMORECEPTION . 9 OLFACTION IN FISH . 11 RATIONALE AND HYPOTHESIS . 14 MATERIALS AND METHODS . 15 SAMPLE COLLECTION AND IDENTIFICATION . 15 EXTERNAL ASSESSMENT . 17 VISUAL ASSESSMENT . 17 SCANNING ELECTRON MICROSCOPY . 18 DATA COLLECTION AND ANALYSIS . 20 RESULTS . 22 MORPHOLOGY OF THE EYE AND OLFACTORY ORGANS. 28 STANDARDIZATION WITH STANDARD LENGTH . 32 vi PRE/POST METAMORPHIC ELOPOMORPHS . 35 DISCUSSION . 38 LITERATURE CITED . 50 vii LIST OF FIGURES Page Figure 1 Chart of larval fish development (Northern anchovy, Engraulis mordax) from end of yolk-sac stage to juvenile stage (Moser and Watson, 2006) . 2 Figure 2 Picture of various distinctive features in leptocephalus larvae (Anibaldi et. al, 2016) . 6 Figure 3 Images of opsin immunohistochemistry of the retinas of ladyfish (A,C,E) and bonefish (B,D,F). 1 = dorsal; 2 = central; 3 = ventral retina regions. Antirod (magenta) and anticone (green, arrowheads) fluorescent stains show the distribution of rods and cones in the retinas of each species (Taylor et al. 2015) . 7 Figure 4 Schematic diagram of olfaction molecular mechanism (Buck and Axel, 1991) . 10 Figure 5 Schematic diagram of general morphology of fish olfactory area (Kasumyan, 2004) . 12 Figure 6 Imagery and drawings of various differences in morphology of fish olfactory area (a) Photo of preserved male anglerfish (Lophiiformes); (b) Electron micrograph from a goldfish (Carrasius auratus); (c) Electron micrograph from European eel (Anguilla anguilla); (ii) all drawings of rosette and lamellae for each corresponding species above (Cox, 2008) . 13 Figure 7 Images of morphometrics taken from an Atlantic menhaden (Brevoortia tyrannus) using Scandium program. (1) Total olfactory area; (2) Eye major and minor axis, posterier nostril major and minor axis, and anterior nostril major and minor axis (left to right); (3) Total eye area, posterior nostril area, anterior nostril area (left to right) . 21 Figure 8 Scanning Electron Micrograph image of an Atlantic croaker (Micropogonias undulatus) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril . 24 viii Figure 9 Scanning Electron Micrograph image of an Atlantic menhaden (Brevoorita tyrannus) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril . 24 Figure 10 Scanning Electron Micrograph image of a Pinfish (Lagodon rhomboides) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril . 25 Figure 11 Scanning Electron Micrograph image of a Speckled worm eel (Myrophis punctatus) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril . 25 Figure 12 Scanning Electron Micrograph image of a Ladyfish (Elops saurus) larva. Magnification = 50x. E = eye; Olf = olfactory pit . 26 Figure 13 Scanning Electron Micrograph image of a Bonefish (Albula vulpes) larva. Magnification = 50x. E = eye; Olf = olfactory pit . 26 Figure 14 Scanning Electron Micrograph image of a Tarpon (Megalops atlanticus) larva. Magnification = 50x. E = eye; Olf = olfactory pit . 27 Figure 15 Mean eye area per species (+/- S.D.). Blue = Leptocephalus larvae; Orange = Non- leptocephalus larvae . 28 Figure 16 Mean olfactory area per species (+/- S.D.). Blue = Leptocephalus larvae; Orange = Non- leptocephalus larvae . 29 Figure 17 Mean (+/- S.D.) ratio of average olfactory area/average eye area in each species; Blue = leptocephalus larvae; Orange = Non- leptocephalus larvae . 30 Figure 18 Mean (+/- S.D.) of standardized eye diameter (% of SL); Blue = Leptocephalus larvae; Orange = Non- leptocephalus larvae . 33 Figure 19 Mean (+/- S.D.) of standardized olfactory pit length (% of SL); Blue = Leptocephalus larvae; Orange = Non- leptocephalus larvae . 34 Figure 20 Scanning Electron Micrograph image of a juvenile bonefish at 20x. SL = 40mm . 36 ix Figure 21 Scanning Electron Micrograph image of a juvenile ladyfish at 20x. 36 SL = 61mm . Figure 22 Scanning Electron Micrograph image of a juvenile pinfish at 20x. SL = 34mm . 37 Figure 23 Image of Muraenesox cinereus consuming squid paste (Mochioka et. al, 1993) . 39 Figure 24 Comparison of head versus body size of a leptocephalus larva versus a larval pinfish . 43 Figure 25 Image of various olfactory rosette types (Kasumyan, 2004) . 46 Figure 26 (A) Scanning Electron Micrograph image of a juvenile Bonefish at 20x; (B) Scanning Electron Micrograph image of the olfactory apparatus at 75x; SL = 40mm . 47 Figure 27 (A) Scanning Electron Micrograph image of a juvenile Ladyfish at 20x; (B) Scanning Electron Micrograph image of the olfactory apparatus at 75x; SL = 61mm . 48 x LIST OF TABLES Page Table 1 Summary of settlement-stage larval fishes examined in this study . 23 Table 2 Matrix showing the results of the KW multiple comparison tests for total eye and olfactory areas. ** = significant at p<0.05. NS = not significant . 31 xi ACKNOWLEDGEMENTS I would like to thank my academic advisor, Dr. Jon Shenker for his guidance and support throughout my master’s program. I would like to thank my remaining committee members, Dr. Turingan, Dr. Hunsucker, and Dr. Webbe for their patience and support. I am so grateful for my friends and labmates, Jake Rennert, Tony Cianciotto, Alex Gering, Jamie Kelly, Mason Thurman, Louis Penrod, and James King, who helped with all the sample collections and sorting. This project would not have been possible without the help and patience of Gayle Duncombe, who taught me all