ResearchOnline@JCU This file is part of the following reference: Tziouveli, Vasiliki (2011) Broodstock conditioning and larval rearing of the marine ornamental white-striped cleaner shrimp, Lysmata amboinensis (de Man, 1888). PhD thesis, James Cook University. Access to this file is available from: http://researchonline.jcu.edu.au/40038/ 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/40038/ Broodstock Conditioning and Larval Rearing of the Marine Ornamental White-striped Cleaner Shrimp, Lysmata amboinensis (de Man, 1888) Thesis submitted by Vasiliki Tziouveli For the degree of Doctor of Philosophy In the Discipline of Aquaculture Within the School of Marine and Tropical Biology James Cook University, QLD, Australia Statement of Access I, the undersigned, the author of this work, understand that James Cook University will make the thesis available for use within the University Library and allow access to users in other approved libraries. I understand that, as an unpublished work, a thesis has significant protection under the Copyright Act and I do not wish to place any further restriction on access to this work. ________________ ______________ Signature Date Vasiliki Tziouveli____________________________ Name ii Statement on sources Declaration I declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institution of tertiary education. Information derived from the published or unpublished work of others has been acknowledged in the text and a list of references is given. _____________________________ _______________ Signature Date Vasiliki Tziouveli____________________________ Name iii Electronic copy declaration I, the undersigned, the author of this work, declare that the electronic copy of this thesis provided to the James Cook University Library is, within the limits of the technology available, an accurate copy of the print thesis submitted. _____________________________ _______________ Signature Date Vasiliki Tziouveli____________________________ Name iv 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 number A1187). _____________________________ ______________ Signature Date Vasiliki Tziouveli______________ Name v Statement on the contribution of others My sincere gratitude to V. Zupo and G. C. Fiedler for reviewing the thesis and helping me improve it through their corrections and suggestions. Publications from the thesis have been co-authored by my supervisors since they provided intellectual and technical guidance and editorial advice. Thanks to the many reviewers for the valuable feedback on the submitted manuscripts. Financial support in the form of stipend and project budget was granted by the Australian Institute of Marine Science and James Cook University (AIMS@JCU). Cairns Marine Aquarium Fish (CMAF) also contributed to the realization of this study by providing the shrimp broodstock gratis. Special thanks to Lyle Squires Jr. and Julian Baggio from CMAF and Matt Kenway from AIMS for making this collaboration successful. Also thanks to my supervisors Mike Hall and Orpha Bellwood for additional funds for international conference attendance and microscopic analysis of extra larval samples, respectively. I am grateful to Wayne Robinson from the University of the Sunshine Coast (USC) and Richard Rowe (JCU) for statistical advice. Also to Robert Lascelles (SGS Australia) and, especially, Ian Brock from the Department of Primary Industries (QDPI) for feed gross composition and fatty acid analysis, respectively. vi Thank you to the JCU histotechnologist Sue Reilly and the team at the JCU Advanced Analytical Centre, specifically Jen Whan, Shane Askew and Kevin Blake for teaching me the required techniques to carry out histology and scanning electron microscopy. I very much enjoyed this part of the PhD. vii Acknowledgements Dedicated to my parents, my sister and my brother, you have always been with me, regardless of distances. A big thank you to my primary supervisor Greg Smith for his invaluable insight, essential guidance and constant support throughout the PhD. Many thanks to the other members of the supervisory team: Mike Hall from AIMS, for his boundless knowledge and constructive comments, Orpha Bellwood from JCU, for her inexhaustible enthusiasm and continuous encouragement, and Chaoshu Zeng from JCU, for the thought-provoking discussions. Special thanks to the manager of the AIMS aquaculture facilities Matt Kenway and the technicians Matthew Salmon, Grant Milton and Justin Hochen for technical advice and practical support during my project years at AIMS. You have taught me a lot! I am grateful to the AIMS transport manager Mick Vaughan and my fellow commuters Barry Tobin, Dave Williams, Stuart Kininmonth, Markus Stowar and Libby Evans for making my life easier and the rides to AIMS fun. I have greatly benefited in meetings with the JCU aquaculture team, especially Malwine Lober and Diyana Noordiyana, thank you for the exchange of ideas and sharing your project experiences. viii Also a thank you to the numerous willing volunteers, all truly appreciated for their help, particularly Sango Talei Mitomi, Muhhamad Azmi Abdul Wahab (or simply Zouzou) and Thibaut Mouthier for being available on short notice and delightful to work with. An extended thank you to my beautiful friends in Australia for being my family away from home and for cheering me up during those “never-ending days”. I am grateful that I got to know you. I will treasure our many laughs! This project has been as much a pleasure as a pain in the… neck. But I would not have had it any other way! ix General Abstract Lysmata amboinensis is a protandric simultaneous hermaphrodite (PSH); individuals develop as males (MPs) and then change to simultaneous hermaphrodites (SHs). PSHs exhibit functions of both males and females during each reproductive cycle. There are a number of important physiological reproductive features that have not been previously studies in this species, including male size at sexual maturity, fecundity of SHs based on body size differences and timing of the sex phase change. To answer the former two questions, MPs were paired with SHs to produce various size combinations. It was found that L. amboinensis MPs were sexually mature at a total length (TL) of 34.0 mm and larval production increased with increasing body length, with large SHs displaying 3 times the larval output of small and medium -sized SHs. To answer the latter question, MPs were exposed to different social situations by being paired with either another MP (of similar or larger size) or with an SH. Indeed, the timing of sex phase change was influenced by the social condition, indicating “environmental sex determination” (ESD). The “default” size at change was 37.1 mm (TL) when MPs were reared alone; when MPs were paired with a larger MP it was the larger MP that changed first to SH, while the partner shrimp changed at a larger size than the “default”. If MPs were paired with similar-sized MPs or with SHs, phase change occurred at a smaller size than the “default”. The observed patterns are discussed in terms of reproductive opportunities for a species occuring in small groups in the wild, referred to as a “low density” spp. Aquaculture production of L. amboinensis has been problematic due to the prolonged duration of larval development, punctuated by periods of high mortality. Broodstock x maturation diets have been shown to affect fecundity and offspring quality. The primary aim of this component of the study was to identify a suitable maturation diet for L. amboinensis from a range of commonly available feeds. The six diets were squid, mussel, adult Artemia, a commercial feed, and combinations of the aforementioned and were fed to L. amboinensis over four reproductive cycles. Broodstock fed the squid-mussel diet lost > ½ of the egg mass during incubation, with decreased larval production, the lowest being 22 larvae. By contrast, broodstock fed adult Artemia retained > 80 % of their eggs; however, hatchability was poor, resulting in low numbers per hatch, ranging from 12 to 51 larvae. The commercial feed yielded high fecundity and larval output. In L. amboinensis, a maturation diet consisting of natural feeds alone results in poor reproductive performance; partial, or complete, replacement with an artificial feed is economically feasible. Lipid enrichment of Artemia to boost their highly unsaturated fatty acid (HUFA) content is a standard procedure to improve diet performance and can be taylored to deliver key nutrients to culture animals. Frozen, lipid enriched adult Artemia were fed to L. amboinensis broodstock to investigate their suitability and elucidate the role of essential