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Highly Streamlined PCR-Based Genetic Identification Of Nova Southeastern University NSUWorks HCNSO Student Theses and Dissertations HCNSO Student Work 1-1-2005 Highly Streamlined PCR-Based Genetic Identification of Carcharhinid Sharks (Family Carcharhinidae) for Use in Wildlife Forensics, Trade Monitoring, and Delineation of Species Distributions Marcy Henning Nova Southeastern University Follow this and additional works at: https://nsuworks.nova.edu/occ_stuetd Part of the Marine Biology Commons, and the Oceanography and Atmospheric Sciences and Meteorology Commons Share Feedback About This Item NSUWorks Citation Marcy Henning. 2005. Highly Streamlined PCR-Based Genetic Identification of Carcharhinid Sharks (Family Carcharhinidae) for Use in Wildlife Forensics, Trade Monitoring, and Delineation of Species Distributions. Master's thesis. Nova Southeastern University. Retrieved from NSUWorks, Oceanographic Center. (117) https://nsuworks.nova.edu/occ_stuetd/117. This Thesis is brought to you by the HCNSO Student Work at NSUWorks. It has been accepted for inclusion in HCNSO Student Theses and Dissertations by an authorized administrator of NSUWorks. For more information, please contact [email protected]. Highly streamlined PCR-based genetic identification of carcharhinid sharks (Family Carcharhinidae) for use in wildlife forensics, trade monitoring, and delineation of species distributions by Marcy L. Henning Thesis submitted to the faculty of the Nova Southeastern University Oceanographic Center in partial fulfillment of the requirements for the degree of Master of Science with a specialty in Marine Biology 2005 Dr. Mahmood Shivji ___________________________________________________ Committee Chairperson Dr. Andrew Rogerson __________________________________________________ Dr. Richard Spieler ____________________________________________________ Table of Contents………………………………………………………………………….i Abstract…………………………………………………………………………………...iv Acknowledgements……………………………………………………………………….vi List of Tables…………………………………………………………………………….vii List of Figures…………………………………………………………………………...viii List of Appendices………………………………………………………………………...x Thesis General Introduction……………………………………………………………..xii Chapter One: Streamlined multiplex PCR identification of ridgeback sharks (Family Carcharhinidae) for law enforcement, fisheries management, and conservation Introduction………………………………………………………………………………..1 Materials and Methods…………………………………………………………………….6 Shark tissue samples………………………………………………………………6 DNA extraction, PCR amplification, and DNA sequencing………………………7 Design and testing of species-specific primers……………………………………7 The Atlantic and global decaplex PCR assays…………………………………….9 Application of assay to identification of shark fins from global trade…………..10 Results…………………………………………………………………………………....11 Characteristics of the ITS2 locus in target species………………………………11 Testing each species-specific primer in the triplex PCR assay…………………..11 Testing the Atlantic and global decaplex PCR assays…………………………...13 Testing unknown confiscated fins and dried shark fins………………………….13 Discussion………………………………………………………………………………..15 Multiplex amplification format……………………………………………………....18 The Atlantic and global decaplex PCR assays……………………………………….20 i Chapter Two: Species-specific primers and single tube, multiplex PCR discrimination of two highly morphologically similar, globally distributed shark species, Carcharhinus leucas and C. amboinensis Introduction……………………………………………………………………………130 Materials and Methods…………………………………………………………….…..134 Shark tissue samples……………………………………………………………134 DNA extraction, PCR amplification, and DNA sequencing……………………135 Design and testing of species-specific primers…………………………………136 The quadraplex PCR assay……………………………………………………..137 Application of quadraplex PCR assay to species identification………………..138 Results………………………………………………………………………………….138 Testing the triplex and quadraplex PCR assays………………………………...138 Testing dried shark fins of unknown species origin……………………………139 Discussion………………………………………………………………………………141 Chapter Three: Species-specific PCR primers for rapid identification of two commercially important carcharhinid shark species, Carcharhinus amblyrhynchos and C. sorrah Introduction…………………………………………………………………………….182 Materials and Methods………………………………………………………………....183 Shark tissue samples……………………………………………………………183 DNA extraction, PCR amplification, and DNA sequencing……………………183 Design and testing of species-specific primers…………………………………184 Application of primers to identification of shark fins from global trade……….186 ii Results………………………………………………………………………………..…186 Testing each species-specific primer in the triplex PCR assay…………………186 Testing unknown market-derived, collected, and confiscated shark fins………187 Discussion……………………………………………………………………………...187 Literature Cited………………………………………………………………………....217 iii Thesis Abstract An increase in worldwide industrialized shark fisheries resulting from the growing demand for shark products, especially shark fins in Asian markets, as well as high levels of bycatch mortality associated with multi-species fisheries have raised suspicions of substantial declines in global shark populations. Due to varying responses by individual species to fishing pressure, it has become necessary to manage sharks on a species-specific basis, which requires the collection of more accurate catch and trade data. The accumulation of such data is hindered, however, by difficulties in identifying species from only landed carcasses, body parts, or fins. This is especially true for many of the commercially important carcharhinid (Family Carcharhinidae) sharks, which are easily confused with one another due to strong morphological similarities and overlapping distributions. The forensic assays described in this thesis expand on a molecular approach developed in the M. Shivji laboratory (Guy Harvey Research Institute, Nova Southeastern University) to resolve these shark species identification issues and allow for rapid and accurate identification of shark body parts. The method involves the development of species-specific primers based on interspecific DNA nucleotide polymorphisms within the nuclear ribosomal internal transcribed spacer 2 (ITS2) locus, and inclusion of these primers in highly streamlined multiplex polymerase chain reaction (PCR) assays. Chapter One describes two separate multiplex PCR assays, each addressing different forensic objectives for U.S. Atlantic and global populations of seven carcharhinid shark species, known as the ridgeback sharks: night (Carcharhinus iv signatus), dusky (Carcharhinus obscurus), Caribbean reef (Carcharhinus perezi), sandbar (Carcharhinus plumbeus), bignose (Carcharhinus altimus), silky (Carcharhinus falciformis), and tiger (Galeocerdo cuvier). An assay is described in Chapter Two for distinguishing the bull (Carcharhinus leucas) and Java (Carcharhinus amboinensis) shark, carcharhinid species that display high levels of morphological similarity as well as similar habitat preference and distributions. The development of species-specific primers for identification of two additional carcharhinid shark species found in international fin markets, gray reef (Carcharhinus amblyrhynchos) and spottail (Carcharhinus sorrah), is detailed in Chapter Three. All of the assays and primers presented here have proven effective for various applications in domestic and international fisheries law enforcement, shark species management, and conservation. The efficient, economical, and simple basis of the assay lends itself to widespread routine application in practical contexts, such as collection of species-specific shark catch and trade data and clarification of the extent of species distributions. Further confirmation of the utility of this assay on a global scale is found in the success of this approach and individual primers for identifying dried shark fins from the Hong Kong fin market, confiscated fins from the U.S., South Africa, Guam, and Palau, and unknown identity samples from a Madagascar fishery. v Acknowledgements First, I would like to thank those who provided samples for the shark species that were the focus of this study: D. Abercrombie, G. Adkinson, L. Beerkircher, D. Chapman, S. Clarke, G. Cliff, C. Conrath, P. Doukakis, K. Draper, K. Duncan, R. Garla, B. Gervelis, M. Grace, D. Grubbs, E. Heist, A. Henderson, E. Jones, J. Levesque, M. Liu, Louisiana State University, R. Martin, Mote Marine Laboratory, L. Natanson, NMFS personnel, L. Noll, D. O’Malley, C. Oravetz, R. Pillans, D. Reid, B. Riegl, M. Shivji, B. Wetherbee, and F. Young. Also, thanks to the NOAA Office of Law Enforcement for collaborating with us on various confiscated fins cases. Without these people and their generosity with these samples, I would have had nothing to work with. Many, many thanks to my major professor, Dr. Mahmood Shivji, for giving me a chance in his laboratory and helping me to realize my true interest in genetics and molecular biology. His encouragement and belief in me always helped me tremendously when times got rough, and I will always be appreciative of his continued support and friendship. To the other two-thirds of my thesis committee, Dr. Andrew Rogerson and Dr. Richard Spieler, I wish to thank them for assistance with my project and for supporting me during my graduate studies at the Oceanographic Center. Since I spent most of my time at the Oceanographic Center as the clerical assistant by day and lab rat by night, I wish to thank the faculty and staff of the NSU Oceanographic Center, especially Dr. Richard Dodge, Dr. Andrew Rogerson,
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