ResearchOnline@JCU This file is part of the following reference: Shao, Luchang (2016) Development of larval fish rearing techniques and nutrient requirements for the green mandarin, Synchiropus splendidus: a popular marine ornamental fish. PhD thesis, James Cook University. Access to this file is available from: http://researchonline.jcu.edu.au/47308/ The author has certified to JCU that they have made a reasonable effort to gain permission and acknowledge the owner of any third party copyright material included in this document. If you believe that this is not the case, please contact [email protected] and quote http://researchonline.jcu.edu.au/47308/ Development of larval fish rearing techniques and nutrient requirement for the green mandarin, Synchiropus splendidus: a popular marine ornamental fish Thesis submitted by Luchang Shao (MSc) in September 2016 For the degree of Doctor of Philosophy In the College of Marine and Environmental Science James Cook University Declaration on Ethics The research presented and reported in this thesis was conducted within the guidelines for research ethics outlined in the National Statement on Ethics Conduct in Research Involving Human (1999), the Joint NHMRC/AVCC Statement and Guidelines on Research Practice (1997), the James Cook University Policy on Experimentation Ethics Standard Practices and Guidelines (2001), and the James Cook University Statement and Guidelines on Research Practice (2001). The proposed research methodology received clearance from the James Cook University Experimentation Ethics Review Committee. Approval numbers: A1851; Principal investigator: Luchang Shao; Finish date: September 30, 2015 i Statement of contribution of others Financial support for this study was provided by Graduate Research School of James Cook University, JCU Postgraduate Research Scholarship. Additional financial support was provided by Graduate Research School of James Cook University for being a recipient of the Graduate Research Scheme during my PhD candidature. Both of my supervisors, A/Prof. Chaoshu Zeng and Dr. Igor Pirozzi, provided excellent academic guidance throughout my experiments and scientific and editorial support. Technical staff of Marine and Aquaculture Research Facility Unit (MARFU), Ben Lawes, Andrew Thompson and Simon Wever provided their assistance in building broodstock tanks and supplying the stock cultures for microalgae species. Jonathan Moorhead helped to build the larval culturing vessels and the recycling system. Fahad Alajmi and Kien Nguyen helped me in the microalgae and copepod maintenance. All biochemical analyses were conducted in the College of Marine and Environmental Science of James Cook University and the Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University using their facilities. Chapter Details of publication(s) on Nature and extent of the which chapter is based intellectual input of each author and their affiliations Shao L, Zeng C (manuscript ready Shao L1 Execution of project, to submit) Insights into the writing and editing importance of copepods as larval Zeng C1 Project conception and ii 3 prey of Synchiropus splendidus: design, supervision and editing effects of copepod density, rotifer co-feeding, larval ingestion rate and fatty acid composition. Shao L, Zeng C (manuscript ready Shao L1 Execution of project, to submit) Effects of timing for writing and editing transition of prey from copepods Zeng C1 Project conception and 4 to rotifers on larval survival, design, supervision and editing growth, development, feeding behavior and efficiency of green mandarin fish, Synchiropus splendidus. Shao L, Zeng C (manuscript ready Shao L1 Execution of project, to submit) Identifying the suitable writing and editing time for introducing Artemia Zeng C1 Project conception and 6 nauplii and subsequently enriched design, supervision and editing metanauplii in either live or dead form for larval rearing of green mandarin fish, Synchiropus splendidus. iii Shao L, Zeng C, Cheng Y Shao L1 Execution of project, (manuscript ready to submit) The writing and editing effects of graded dietary DHA Zeng C1 Project conception and 7 (22:6n-3) levels on growth, design, supervision and editing survival and tissue fatty acid Cheng Y2 Project conception and profile of the post-settlement design, technical advice on fatty green mandarin fish, Synchiropus acid analysis splendidus. Shao L, Zeng C, Cheng Y Shao L1 Execution of project, (manuscript ready to submit) The writing and editing effects of graded dietary ARA Zeng C1 Project conception and 8 (20:4n-6) levels on growth, design, supervision and editing survival and tissue fatty acid Cheng Y2 Project conception and profile of the post-settlement design, technical advice on fatty green mandarin fish, Synchiropus acid analysis splendidus. Affiliations 1 Centre for Sustainable Tropical Fisheries and Aquaculture, College of Marine and Environmental Science, James Cook University, Townsville, QLD 4811, Australia 2 Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, No 999 Huchenghuan Road, Lingang New District, Shanghai 201306, China iv Acknowledgements A long list of people has continuously supported and helped me during the past four years of my PhD journey and has contributed to make it what it is today. Firstly and foremost, I wish to thank my primary supervisor A/Prof. Chaoshu Zeng for accepting me as one of his research students and providing me with excellent guidance throughout the years. He was always ready to assist when required and help to improve my research ability in all aspects, especially in the academic writing. I am also grateful to my associate supervisor Dr. Igor Pirozzi for providing me with valuable insights in lipid nutrition of marine fish larvae. He was also always there for me and offered continuous support, advice and guidance over the course of my PhD. I would like to thank all the staff at the Marine and Aquaculture Facility Unit (MARFU), James Cook University: Ben Lawes, Andrew Thompson and Simon Wever for all their technical help in building the lab and maintaining the culture system. Sincere thanks are also given to Prof. Yongxu Cheng from Shanghai Ocean University (SHOU) for providing the analytical chemicals and equipment at SHOU. I am very appreciative to all the students and colleagues that I have met at James Cook University: Laura Ezquerro, Jonathan Moorhead, Fahad Alajmi, Kien Nguyen and Changkao Mu. Thank you for offering hands, suggestions and encouragement throughout the years. I am also greatly indebted to my friends outside of the academic fields, thank you for coloring my PhD life and reminding v me that academic world is not the only world. Lastly but not least, I would like to thank my parents, Xingguo Shao and Qingying Lu have enormously supported me to seek my dream in Australia and always encouraged me when I felt worried, depressed and exhausted. I could not complete my PhD study without their continuous support. Dad and Mom, I love you! This thesis is dedicated to all these lovely people. vi Abstract The green mandarin fish, Synchiropus splendidus (Herre, 1927) is a small, brilliantly coloured benthic marine fish distributed in the tropical Pacific and Indian Oceans. It is within the group of fish known as dragonets (Family: Callionymidae) and is among the most desirable species to marine aquarium hobbyists worldwide. Unfortunately, the current supply of S. splendidus for the aquarium trade comes solely from the wild. Moreover, it has been observed that most specimens on sale are males, suggesting probably selective harvesting from the wild populations. As a result, the captive breeding of S. splendidus is urgently required as an alternative to ensure a sustainable supply of this popular species for the aquarium trade to reduce the pressure on its natural populations. S. splendidus is a pelagic-spawner and has been considered as a relatively hard-to-breed species due to its adults generally feeding on live small crustaceans, hence this species is difficult to maintain under captive condition. In addition, there is limited knowledge on its larval feeding habitat. Newly hatched larvae of S. splendidus is among the smallest larvae of marine fish, on average only 1.5 mm. The high vulnerability of pre-feeding larvae to handling stress and advent environmental conditions make their rearing even more difficult. A series of experiments were conducted to improve captive breeding techniques for S. splendidus, which could also serve as a model species for ornamental marine pelagic-spawners. The present thesis consists of 9 chapters: following the first general introduction chapter (Chapter 1), the second chapter describes general materials and methods used (Chapter 2). The subsequent 6 data vii chapters can be largely grouped into three main themes: 1) Chapter 3, 4 and 5 focused on the first feeding preys and culturing regimes for newly hatched and early larvae of S. splendidus, and investigated the underlying mechanisms for the superior performance of copepods as live prey from the perspectives of behavioral and lipid nutrition. 2) Chapter 6 then focused on the strategy and best timing for larval prey transition from copepods/rotifers to Artemia nauplii, and subsequently to larger enriched metanauplii during the critical metamorphosis period of S. splendidus. 3) Chapters 7 and 8 finally focused on optimizing the growth rate of post-settlement S. splendidus by their feeding with