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Conopeptide Production Through Biosustainable Snail Farming A Conopeptide Production through Biosustainable Snail Farming A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MOLECULAR BIOSCIENCES AND BIOENGINEERING DECEMBER 2012 By Jeffrey W. Milisen Thesis Committee: Jon-Paul Bingham (Chairperson) Harry Ako Cynthia Hunter Keywords: Conus striatus venom variability Student: Jeffrey W. Milisen Student ID#: 1702-1176 Degree: MS Field: Molecular Biosciences and Bioengineering Graduation Date: December 2012 Title: Conopeptide Production through Biosustainable Snail Farming We certify that we have read this Thesis and that, in our opinion, it is satisfactory in scope and quality as a Thesis for the degree of Master of Science in Molecular Biosciences and Bioengineering. Thesis Committee: Names Signatures Jon-Paul Bingham (Chair) ___________________________ Harry Ako ___________________________ Cynthia Hunter ___________________________ ii Acknowledgements The author would like to take a moment to appreciate a notable few out of the army of supporters who came out during this arduously long scholastic process without whom this work would never have been. First and foremost, a “thank you” is owed to the USDA TSTAR program whose funds kept the snails alive and solvents flowing through the RP-HPLC. Likewise, the infrastructure, teachings and financial support from the University of Hawai‘i and more specifically the College of Tropical Agriculture and Human Resources provided a fertile environment conducive to cutting edge science. Through the 3 years over which this study took place, I found myself indebted to two distinct groups of students from Dr. Bingham’s lab. Those who worked primarily in the biochemical laboratory saved countless weekend RP-HPLC runs from disaster through due diligence while patiently schooling me on my deficiencies in biochemical processes and techniques. Even more, I would like to thank the students assigned to Dr. Bingham’s aquaculture facility who had to work with me directly during this time, not only with regards to this study but also in the foundational design and building of the aquaculture facility. They were often asked to spend a full afternoon from their other responsibilities in my absence to feed my snails, a process that can be excruciating when the snails aren’t terribly hungry. This was in addition to their daily upkeep tasks, which often required precious weekend commitments as well. I know the sacrifices you all made and I am sincerely grateful. For their slimy donations, I’d like to thank Hank Lynch and J. J. Jackson because, try as I might, I’m still the world’s worst shell collector. Without your contributions, I would have been studying ten empty tanks full of seawater. This brings me to the tireless efforts put forth by the inexhaustible Dr. Jon-Paul Bingham who somehow manages to meet with each of his students every week while teaching the university’s flagship biochemistry course and still figures out a way to make himself available for the odd question of protocol. The opportunities this project afforded me along with the guidance and lessons have returned invaluable life experiences that will follow me for the rest of my life. Finally I would like to take a brief moment to show some gratitude to my personal support team. For all the dinners you delivered when I was stuck at school late at night and for providing the at-home logistical support to keep my demeanor and appearance appropriate for the rest of the world, I will always be indebted to Melanie Kosaka. And finally, to my dad who was a single parent wrestling with two maturing sons and now dutifully serves the role of moral supporter in the face of impossible times. Thank you. iii ABSTRACT An area of intensive focus and research has been in the study of conopeptides isolated from cone snail venom. These complex peptide venoms contain between 50 and 100 different bioactive substances, many of which can be utilized for a variety of anthropogenic needs. Bioprospecting from this limited marine resource has caused ecological concerns, echoing that selected Conus species should be listed under CITES to avoid their demise. Utilizing captive husbandry techniques to promote biosustainable methods for venom collection, links were established between venom volume and snail size, venom volume and seasonality, venom volume and time in captivity, lunar phase and feed rate and lunar phase and venom diversity to maximize venom peptide diversity and output from cone snails as a valuable pharmacological source. iv Table of Contents Acknowledgements ......................................................................................................................... iii ABSTRACT .......................................................................................................................................... iv Table of Figures ............................................................................................................................... vii List of Tables ..................................................................................................................................... xi INTRODUCTION ........................................................................................................................ 1 Hawai‘ian BiogeograpHy ................................................................................................................ 1 The Ecology of Conus striatus in Hawai‘i .................................................................................. 1 Cone Snail Anatomy and Physiology .......................................................................................... 2 Larval Development ........................................................................................................................ 3 Feeding Behavior .............................................................................................................................. 4 Venom Delivery ................................................................................................................................. 5 OtHer Venoms .................................................................................................................................... 6 Intersexual Variations in Venom Composition ...................................................................... 7 Conopeptides of Conus striatus .................................................................................................... 7 Conus Venom Sources .................................................................................................................... 12 Venom Variation in Conus ........................................................................................................... 13 AntHropogenic Uses for Conopeptides .................................................................................... 14 Biosustainability ............................................................................................................................. 15 Moon PHase ....................................................................................................................................... 15 Taxonomy of Conus striatus ........................................................................................................ 16 HYPOTHESES ........................................................................................................................... 18 General HypotHeses ....................................................................................................................... 18 Specific HypotHeses ....................................................................................................................... 18 Reproduction ................................................................................................................................................. 18 Snail Size .......................................................................................................................................................... 19 Sex ....................................................................................................................................................................... 19 Death .................................................................................................................................................................. 19 Pill Diet .............................................................................................................................................................. 19 Morphological Variants ............................................................................................................................. 20 Phenology-Seasonality ............................................................................................................................... 20 Moon Phase ..................................................................................................................................................... 20 Time in Captivity .......................................................................................................................................... 20 METHODS ................................................................................................................................. 21 Housing .............................................................................................................................................
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