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Exploring the Potential of the Sea Cucumber Cucumaria Frondosa As

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Exploring the Potential of the Sea cucumber Cucumaria frondosa as an Aquaculture Species By © Bruno Lainetti Gianasi A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Ocean Sciences Memorial University of Newfoundland St. John’s, Newfoundland and Labrador, Canada November 2018 Abstract Sea cucumbers are a luxury seafood in China and the increasing demand is driving aquaculture initiatives towards new species worldwide. Cucumaria frondosa has been commercially harvested for decades in North America and has also been identified as a potential candidate for aquaculture. So far, captive-breeding methods have focused on tropical/temperate deposit-feeding species with planktotrophic development (small eggs, feeding larvae); however, C. frondosa is a cold-water suspension-feeder with lecithotrophic development (large yolky eggs, non-feeding larvae). The aim of this study was to explore the reproductive biology, larval development, and juvenile ecology of C. frondosa to identify optimum conditions for culture. In an experimental study, adults kept in ambient environmental conditions showed the best reproductive output and embryo survival relative to those in warmer water, advanced photoperiod, and constant light or dark conditions. An increase in food concentration did not enhance the reproductive output. When methods used to trigger spawning and artificially induce oocyte maturation were investigated, live phytoplankton at 1 x 105 cell ml-1 was the most successful technique; moreover, among a handful of chemicals tested, only Dithiothreitol (DTT) at 10-1 M induced significantly more oocytes to ovulate than the control (seawater), although eggs remained unfertilizable. These experiments led to the discovery of embryonic fusion and the formation of chimaeras (individuals composed of genetically distinct types of cells). Fusion occurred only among hatched blastulae and occurred in a maximum of 9% of the propagule population, generating individuals up to 5 times larger than singletons. Finally, development and ii behaviour of juveniles assessed from settlement to 21 months of age revealed that newly- settled juveniles had low tolerance to light and water flow, fed on deposited material, and preferred rocky substrates and black or red backgrounds. With age, juveniles grew ramified tentacles, shifted to suspension feeding, developed greater tolerance to light and water flow, and showed a preference for coralline algae and red backgrounds. The findings presented here provide important advances to optimize gonad development and egg quality in captive broodstock of C. frondosa, to induce or enhance spawning and fertilization, as well as to elucidate the optimal conditions for embryos and juveniles. This work presents a pilot-scale assessment of commercial production in C. frondosa, with potential applicability to similar cold-water suspension-feeding species. iii Acknowledgement I would like to thank my supervisor Annie Mercier for her guidance, patience, encouragement, and great dedication throughout both my MSc and PhD; all these years have been an amazing time. I would also like to thank Jean-Francois Hamel for his motivation, passion, countless hours of hard work, and guidance which always helped me keep focused. I thank Phil Barnes (Fogo-Island Co-operative Society) and Laura C. Halfyard (Sunrise Fish Farms Inc) for being supportive industry partners, providing sea cucumbers and information about harvests and water parameters at the farm sites. I also appreciated the insightful comments and encouragement of my committee members, Cyr Couturier and Iain McGaw, which helped approach my research questions from different angles. Many thanks to my friends Danny Boyce, Jennifer Monk, Denise Tucker, Jessica Fry, Kelsie Jeannot, and Chantal Gionet for giving me opportunities to learn about aquaculture of finfish and shellfish and for their invaluable assistance with experimental logistics. I thank my fellows labmates for all the fun moments even during long days and late (super late) nights in the lab. Special thanks to Matt Osse, Jiamin Sun, Michelle Valliant, and MacGregor Parent for helping with measurements, dissections, gamete collection and numerous counts under the microscope. This research was funded by the Research and Development Corporation of Newfoundland and Labrador (RDC), the Canadian Centre for Fisheries Innovation (CCFI) iv and the Department of Fisheries and Aquaculture of Newfoundland and Labrador (DFA). I was the recipient of an RDC Ocean Industries Student Research Award (OISRA). Finally, I am very grateful for the constant support and encouragement of my family and friends throughout my graduate studies. v Table of Contents Abstract ............................................................................................................................... ii Acknowledgement .............................................................................................................. iv Table of Contents ................................................................................................................ vi List of Tables ....................................................................................................................... x List of Figures ..................................................................................................................... xi List of Appendices .......................................................................................................... xvii Co-authorship Statement ................................................................................................... xix Chapter 1. General Introduction........................................................................................... 1 1.1 From traditional medicine to aquaculture .................................................................. 2 1.2 Cucumaria frondosa as a candidate for aquaculture .................................................. 4 1.3 Main objectives and thesis structure .......................................................................... 7 1.4 References ................................................................................................................ 11 Chapter 2. Environmental Control and Manipulation of Gametogenesis and Spawning: Egg Quality, and Embryo Survival in the Sea Cucumber Cucumaria frondosa ............... 18 2.1 Abstract .................................................................................................................... 19 2.2 Introduction .............................................................................................................. 21 2.3 Material and methods ............................................................................................... 25 2.3.1 Sea cucumber collection .................................................................................... 25 2.3.2 Experimental design .......................................................................................... 25 2.3.3 Monitoring and data collection .......................................................................... 29 2.3.4 Data analysis ...................................................................................................... 32 2.4 Results ...................................................................................................................... 33 2.4.1 Health condition, reproductive output, and spawning ....................................... 33 2.4.2 Egg/embryo quality and survival ....................................................................... 38 2.5 Discussion ................................................................................................................ 40 2.6 Conclusion ................................................................................................................ 45 vi 2.7 Acknowledgments .................................................................................................... 45 2.8 References ................................................................................................................ 46 2.9 Tables ....................................................................................................................... 52 2.10 Figures .................................................................................................................... 55 2.11 Supplementary Material ......................................................................................... 66 Chapter 3. Triggers of Spawning and Oocyte Maturation in the Commercial Sea Cucumber Cucumaria frondosa ......................................................................................... 70 3.1 Abstract .................................................................................................................... 71 3.2 Introduction .............................................................................................................. 73 3.3 Material and Methods ............................................................................................... 78 3.3.1 Collection of broodstock and holding tanks ...................................................... 78 3.3.2 Spawning induction ........................................................................................... 79 3.3.3 Artificial induction of oocyte maturation and fertilization in vitro ................... 83 3.3.4 Statistical analysis .............................................................................................
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