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REDUCING POST-BLEEDING MORTALITY of HORSESHOE CRABS (Limulus Polyphemus) USED in the BIOMEDICAL INDUSTRY

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REDUCING POST-BLEEDING MORTALITY of HORSESHOE CRABS (Limulus Polyphemus) USED in the BIOMEDICAL INDUSTRY REDUCING POST-BLEEDING MORTALITY OF HORSESHOE CRABS (Limulus polyphemus) USED IN THE BIOMEDICAL INDUSTRY By Lenka Hurton Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN FISHERIES AND WILDLIFE SCIENCES Approved: Dr. Jim Berkson Advisory Chair Dr. Eric Hallerman Committee Member Dr. Stephen Smith Committee Member December 2003 Blacksburg, Virginia Keywords: mortality, blood volume, hemolymph, amebocyte, Limulus polyphemus, horseshoe crab Copyright 2003, Lenka Hurton REDUCING POST-BLEEDING MORTALITY OF HORSESHOE CRABS (Limulus polyphemus) USED IN THE BIOMEDICAL INDUSTRY by: Lenka Hurton Jim Berkson, Chair Fisheries and Wildlife Sciences (ABSTRACT) The biomedical industry bleeds horseshoe crabs (Limulus polyphemus) to produce a product from the blood cells that is used to test the sterility of medical and pharmaceutical products. This study examined the effects of blood extraction on the survival of horseshoe crabs and performed a preliminary investigation into amebocyte maintenance in vitro. Hemolymph volume of L. polyphemus was estimated over a representative size range of adults. Hemolymph volume expressed as a percentage of wet body weight was 25 ± 2.2% (mean ± S.D.) for males and 25 ± 5.1% for females. Relationships of hemolymph volume to weight (P = 0.0026, r2 = 0.8762), hemolymph volume to prosomal width (P < 0.0001), and hemolymph volume to inter- ocular width (P < 0.0001) were identified. Mortality associated with blood extraction was evaluated for horseshoe crabs bled 0, 10, 20, 30, and 40% of their estimated hemolymph volume (unstressed group, N = 200). Mortality associated with the same bleeding levels was evaluated in horseshoe crabs that underwent simulated transport and handling procedures of the biomedical industry’s bleeding process (stressed group, N = 195). Mortality rates of the unbled crabs were not significantly different between the stressed group and unstressed group. Of the bled animals, there was a higher (8.3%) mortality rate in the stressed group, than that (0%) in the unstressed group (P < 0.0001). Within the stressed group, mortality was significantly associated with bleeding (P = 0.0088), and overall mortality increased with increasing levels of blood extraction (P = 0.006, r2 = 0.994). The recovery of hemolymph volume over a 12-day period was monitored (N = 45). Total hemolymph protein levels in the 10, 20, 30, and 40%-bled groups all demonstrated decreased levels between days 0 and 3 (P < 0.0001) post-bleeding. Horseshoe crab serum and a variety of standard invertebrate and insect cell culture media were evaluated for their effects on amebocyte morphology and viability after 7 days of maintenance in vitro. Horseshoe crab serum-supplemented cultures had significantly higher cell viability than serum-free cultures (N = 6; P = 0.0147). Significant differences in amebocyte viability were identified among the six insect cell culture media tested (N = 36; P < 0.0001). Amebocytes in Grace’s Insect Medium without serum remained 77.2 ± 5.1% (mean ± S.D.) viable, followed distantly by Grace’s Modified Insect Medium without serum with a viability of 35.1 ± 8.7%. Taking amebocyte morphology and viability into consideration, this study indicated that Grace’s Insect Medium with horseshoe crab serum supplementation was the best candidate for future medium optimization for Limulus amebocyte requirements. Information gained from this study provides guidance on altering biomedical bleeding protocols to decrease horseshoe crab stress and mortality, and advances information on amebocyte culture medium selection, both of which contribute to decreasing the biomedical industry’s impact on the horseshoe crab population. III ACKNOWLEDGEMENTS I thank my advisor, Dr. Jim Berkson, for providing me with the opportunity to work on this project. His unwavering support, constructive advice, and dedicated effort over the course of this project are greatly appreciated. My committee members, Dr. Stephen Smith and Dr. Eric Hallerman, provided valuable guidance and input throughout this project, and their efforts are greatly appreciated as well. This work is a result of research sponsored in part by the NOAA Office of Sea Grant, US Dept of Commerce, under Grant No. NA96RG0025 to the Virginia Graduate Marine Science Consortium and Virginia Sea Grant College Program. Cambrex Bio Science Walkersville, Inc. generously supplied my project with horseshoe crabs and cell culture media. I would like to thank Dr. Ron Berzofsky and all of Cambrex Bio Science Walkersville, Inc. (Cambrex) for their support throughout this project. I would also like to thank Jeffrey Eustler and his boat crew for his patience and support when he supplied us with horseshoe crabs on behalf of Cambrex. Finally, I would like to thank my parents, friends, and fellow graduate students for their encouragement and invaluable assistance with my research. I would especially like to thank Vincent Caruso, Dr. Dave Hata, René Olsen, Michelle Davis, Whitney Grogan, Jay McGhee, Mary Tilton, Alison Williams, and Alison Sasnett for aiding in transporting, tagging, and/or bleeding horseshoe crabs for this study. In particular, Whitney Grogan provided much appreciated advice during the writing process. IV TABLE OF CONTENTS ACKNOWLEDGEMENTS .......................................................................................................IV LIST OF TABLES .................................................................................................................... VII LIST OF FIGURES .................................................................................................................VIII CHAPTER 1 – INTRODUCTION AND JUSTIFICATION.................................................... 1 INTRODUCTION ...................................................................................................................... 1 Commercial Fishery................................................................................................................ 1 Shorebirds and Conservationists ............................................................................................ 2 Biomedical Industry................................................................................................................ 4 PROJECT PURPOSE AND OBJECTIVES............................................................................... 9 REFERENCES ......................................................................................................................... 12 CHAPTER 2 - DETERMINATION OF TOTAL HEMOLYMPH VOLUME IN THE HORSESHOE CRAB ................................................................................................................. 15 ABSTRACT.............................................................................................................................. 15 INTRODUCTION .................................................................................................................... 16 METHODS ............................................................................................................................... 17 Calculations and statistical analyses.................................................................................... 19 RESULTS ................................................................................................................................. 19 DISCUSSION........................................................................................................................... 20 ACKNOWLEDGEMENTS...................................................................................................... 24 REFERENCES ......................................................................................................................... 25 CHAPTER 3 - POST-BLEEDING MORTALITY OF HORSESHOE CRABS .................. 31 ABSTRACT.............................................................................................................................. 31 INTRODUCTION .................................................................................................................... 32 METHODS ............................................................................................................................... 36 Bleeding mortality study 1 .................................................................................................... 37 Bleeding mortality study 2 .................................................................................................... 38 Statistical analyses................................................................................................................ 40 V RESULTS ................................................................................................................................. 40 DISCUSSION........................................................................................................................... 42 ACKNOWLEDGEMENTS...................................................................................................... 45 REFERENCES ......................................................................................................................... 46 CHAPTER 4 - SELECTION OF A CELL CULTURE MEDIUM FOR IN VITRO MAINTENANCE OF LIMULUS AMEBOCYTES................................................................. 53 ABSTRACT.............................................................................................................................
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