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Reproductive Physiology of the Female Blue Crab, Callinectes Sapidus : Spawning Induction and Vitellogenesis

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Reproductive Physiology of the Female Blue Crab, Callinectes Sapidus : Spawning Induction and Vitellogenesis ABSTRACT Title of Document: REPRODUCTIVE PHYSIOLOGY OF THE FEMALE BLUE CRAB, CALLINECTES SAPIDUS : SPAWNING INDUCTION AND VITELLOGENESIS Sarah Elizabeth Bembe Master of Science 2009 Directed By: Dr. J. Sook Chung University of Maryland Biotechnology Institute Center of Marine Biotechnology In aquaculture, spawning is the baseline for production; therefore, the optimization of spawning conditions will directly increase production. The current study aims to optimize spawning conditions for Callinectes sapidus using environmental manipulations of photoperiod and temperature for induction while monitoring the physiological vitellogenin (VtG) levels during ovarian development and maturation. The photothermal manipulations for this study resulted in increased spawning events in 21 oC temperatures (compared to 11 oC and 15 oC) and complete darkness (0L:24D; compared to 8L:16D, 16L:8D, and 24L:0D) while 24L:0D and 11 oC suppressed spawning. When assessing the VtG levels in the hemolymph prior to, during, and after all spawning events, the VtG showed a decrease prior to spawning, and significant VtG activity was seen in 21 oC for all photoperiods. Overall, spawning and vitellogenesis are temperature dependent events with 67% of the females spawning in 21 oC. Photoperiod also has an effect on spawning, but not on vitellogenesis. REPRODUCTIVE PHYSIOLOGY OF THE FEMALE BLUE CRAB, CALLINECTES SAPIDUS : SPAWNING INDUCTION AND VITELLOGENESIS By Sarah Elizabeth Bembe Thesis submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Master of Science 2009 Advisory Committee: Assistant Professor Dr. J. Sook Chung, Chair Dr. Anson H. Hines Dr. Allen R. Place Dr. Yonathan Zohar © Copyright by Sarah Elizabeth Bembe 2009 ACKNOWLEDGEMENTS I would like to thank everyone for their support and help throughout my studies and thesis writing. Especially, NOAA Chesapeake Bay Program for providing funding for this project (NOAA award #NA17FU2841). Special thanks need to be given to: • My advisor, J. Sook Chung, for teaching me how to be a molecular biologist, being patient with me, and allowing me to see the positive in the negative. • My committee members for their input and questions which kept me on track. • Steve Rodgers, Eric Evans, Chris Tollini, Daria Pedora, and Joy Harris for their help building, caring for, and fixing the tank system. • Oded Zmora for un-officially serving on my committee and providing useful hatchery information and advice. • Ralph Yates for allowing me to board his boat (Critter Gitter) and selectively collect crabs from his harvest multiple times. • Rebecca Cluster, my partner in crime, for feeding the crabs and helping me on the Critter Gitter. • Andrea Allman for past hatchery information and crab rearing advice. • Stephanie Grap and Katherine Rhea for feeding the crabs on short notice. • Nilli Zmora, to whom I am indebted, for all of her help, patience, and time dealing with the ELISAs. • Sirinart Techa for listening to my woes and being a good friend. • Naoaki Tsuisui for teaching me the ins-and-outs of northern blotting and cloning and designing the under-the-sand filtration setup. • John Stubblefield for being the ‘King of Everything’. • The members of the National Aquarium of Baltimore water quality lab for providing useful water quality information and readings. • My mother and godmother for reading my thesis multiple times and providing useful corrections. • My family for keeping me grounded. • And finally, my husband, for trying to understand the commitment I had to make to my research, making me laugh, and loving me unconditionally. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................. ii TABLE OF CONTENTS .................................................................................................. iii LIST OF TABLES ............................................................................................................ v LIST OF FIGURES .......................................................................................................... vi ABBREVIATIONS ......................................................................................................... vii SPECIES ...................................................................................................................... viii CHAPTER 1: I NTRODUCTION ........................................................................................ 1 ASPECTS OF AQUACULTURE ............................................................................ 1 CRUSTACEAN AQUACULTURE ......................................................................... 2 CALLINECTES SAPIDUS AQUACULTURE ....................................................... 11 CRUSTACEAN REPRODUCTION AND VITELLOGENESIS .......................... 14 PROJECT OBJECTIVES AND ORGANIZATION OF THE STUDY ................. 22 CHAPTER 2: E NVIRONMENTAL MANIPULATIONS TO INDUCE SPAWNING ................ 24 INTRODUCTION .................................................................................................. 24 MATERIALS AND METHODS ............................................................................ 28 Animal Collection ............................................................................................... 28 Acclimation of Wild Crabs .................................................................................. 30 Photoperiod and Temperature Manipulations .................................................... 30 Spawning Observations ...................................................................................... 35 Biopsy of Ovarian Tissue .................................................................................... 36 Long-term Hatchery Data ................................................................................... 36 Statistical Analysis .............................................................................................. 37 RESULTS ............................................................................................................... 37 Temperature vs. Photoperiod .............................................................................. 37 Ovarian and Embryo Development .................................................................... 39 Spawning Data Comparison ............................................................................... 42 DISCUSSION ......................................................................................................... 44 Photothermal Manipulations .............................................................................. 44 Ovarian and Embryo Development .................................................................... 48 Spawning Data Comparison ............................................................................... 50 CONCLUSION ....................................................................................................... 52 CHAPTER 3: H EMOLYMPH VITELLOGENIN LEVELS DURING OVARIAN DEVELOPMENT ..................................................................................................................................... 53 INTRODUCTION .................................................................................................. 53 MATERIALS AND METHODS ............................................................................ 55 Animal Collection and Care ............................................................................... 55 Hemolymph Sampling ......................................................................................... 57 Vitellin Purification ............................................................................................ 57 Affinity Purification of Anti-VT ........................................................................... 59 Rabbit Anti-VT Serum ...................................................................................... 59 Column Preparation .......................................................................................... 59 VT Purification ................................................................................................. 60 Affinity Purificatin of Anti-VT ......................................................................... 60 Competitive ELISA .............................................................................................. 62 iii Coating Plate ..................................................................................................... 62 Standard and Sample Preparation ..................................................................... 62 Assay ................................................................................................................. 63 Protein Quantification ........................................................................................ 64 Statistical Analysis .............................................................................................. 64 RESULTS ............................................................................................................... 65 Competitive ELISA .............................................................................................. 65 Non-spawned Females ...................................................................................... 65 Spawned Females.............................................................................................. 66 Temperature
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