REPRODUCTIVE DEVELOPMENT OF FEMALE BONEFISH (ALBULA SPP.) FROM THE BAHAMAS by Cameron Alexander Luck A Thesis Submitted to the Faculty of The Charles E. Schmidt College of Science In Partial Fulfilment of the Requirements for the Degree of Master of Science Florida Atlantic University Boca Raton, Florida December 2018 Copyright 2018 by Cameron Luck ii ACKNOWLEDGEMENTS I sincerely appreciate the guidance of my advisor Dr. Matthew Ajemian for his continued support throughout this endeavor as well as the input from my committee members: Dr. Sahar Mejri, Dr. Aaron Adams, and Dr. Paul Wills. I am extremely grateful for the collaboration and support of Justin Lewis. Field collections would have been impossible without his local knowledge, willingness to be available, passion for bonefish, and incredible work ethic. I am also grateful for the words of wisdom and comedic relief often provided by fellow graduate students, lab members, and interns of the Fish Ecology and Conservation Lab: Grace Roskar, Breanna Degroot, Steve Lombardo, Rachel Shaw, Mike McCallister, Alex Allen, and many others. This project was financially supported by Bonefish & Tarpon Trust (BTT) and National Fish and Wildlife Foundation (NFWF). We are grateful for the lodging and water access provided by East End Lodge, Andros South, and Hank’s Place. The experimental protocol for this study received approval from Florida Atlantic University’s Institutional Animal Care and Use Committee (Animal Use Protocol #A16-34). iv ABSTRACT Author: Cameron Luck Title: Reproductive Development of Female Bonefish (Albula spp.) from the Bahamas Institution: Florida Atlantic University Thesis Advisor: Dr. Matthew J. Ajemian Degree: Master of Science Year: 2018 Bonefish (Albula spp.) support an economically important sport fishery, yet little is known regarding the reproductive biology of this genus. Analysis of oocytes histology and sex hormone levels was conducted on wild female bonefish sampled during and outside the spawning season in Grand Bahama, Central Andros, and South Andros, The Bahamas to assess reproductive state. Bonefish are commonly found along shallow water flats, or in pre-spawn aggregations (PSA) during spawning months. 17β-estradiol levels suggest vitellogenic consistency between habitats. However, fish are more reproductively developed at PSA based on the occurrence of larger, more prevalent vitellogenic oocytes and evidence of final maturation. Variability in hormone levels and spawning readiness existed between Grand Bahama and Andros PSAs, suggesting peak spawning may differ v by region. Findings from this study will contribute baseline data to the captive bonefish restoration project at Harbor Branch Oceanographic Institute and to the limited ecological data regarding bonefish reproduction. vi REPRODUCTIVE DEVELOPMENT OF FEMALE BONEFISH (ALBULA SPP.) FROM THE BAHAMAS LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES .............................................................................................................x BACKGROUND .................................................................................................................1 Spawning strategies in the marine environment ............................................................1 Historic Anthropogenic Impact on Aggregate Spawners ..............................................3 Physiological Indicators of Spawning in Marine Fishes ...............................................4 Project Objectives ..........................................................................................................8 CHAPTER 1 ........................................................................................................................9 INTRODUCTION .........................................................................................................9 METHODS ..................................................................................................................12 Initial sample collection and preservation ............................................................12 Histological preparation of oocytes.......................................................................13 Sex-hormone plasma level determination ..............................................................14 Statistical analysis .................................................................................................15 RESULTS ....................................................................................................................16 Histology ................................................................................................................16 Sex-hormone concentration ...................................................................................16 I. Seasonal differences...................................................................................16 vii II. Flats vs. PSA ..............................................................................................17 DISCUSSION Seasonal differences...............................................................................................17 Flats vs. pre-spawn aggregation............................................................................18 CHAPTER 2 ......................................................................................................................23 INTRODUCTION .......................................................................................................23 METHODS ..................................................................................................................27 Sample collection ...................................................................................................27 Histological preparation of oocytes.......................................................................28 Reproductive phase classification ..........................................................................28 Sex-hormone plasma level determination ..............................................................29 Statistical analysis .................................................................................................31 RESULTS ....................................................................................................................32 Histology ................................................................................................................33 Sex hormone concentrations ..................................................................................33 I. Seasonal differences...................................................................................33 II. Spatial differences ......................................................................................33 III. Reproductive phase classification ..............................................................34 DISCUSSION ..............................................................................................................35 Seasonal development ............................................................................................35 Spatial trends .........................................................................................................36 CONCLUSION AND FUTURE DIRECTIONS ...............................................................41 APPENDICES ..................................................................................................................44 viii APPENDIX A. FIGURES ...........................................................................................45 APPENDIX B. TABLES .............................................................................................58 REFERENCES ..................................................................................................................61 ix LIST OF FIGURES Figure 1. Map showing sampling locations for both flats and pre-spawn aggregation (PSA) sites during spawning (February, March, and April 2017) and non- spawning (September 2017) months of bonefish (Albula vulpes) in Grand Bahama Island, Bahamas ....................................................................................45 Figure 2. Micrographs of histologically sectioned oocytes sample collected from a wild female bonefish (Albula vulpes) on a shallow water flat during full moon (April 2017) ..............................................................................................46 Figure 3. Proportional occurrence for each of the three oocyte stages: late vitellogenic (LV), cortical alveolar (CA), primary growth (PG) at the two sites (flats and pre-spawn aggregation [PSA]) sampled during the spawning month of April 2017.....................................................................................................................47 Figure 4. Monthly plasma concentrations of 17β–estradiol and Testosterone (mean SEM) from bonefish (Albula vulpes) females sampled in the flats during spawning (February and April 2017) and non-spawning (September 2017) months (mean SEM) ........................................................................................48 Figure 5. Plasma concentrations of 17β–estradiol and testosterone (mean SEM) at flats and pre-spawn aggregation (PSA) sites in female bonefish (Albula vulpes) sampled during three spawning months (February, March, and April 2017) ..........................................................................................................49 x Figure