HISTAMINE – A NATURALLY OCCURRING SETTLEMENT CUE FOR LARVAE OF THE AUSTRALIAN SEA URCHIN Holopneustes purpurascens Rebecca L. Swanson A thesis submitted to the University of New South Wales for the degree of Doctor of Philosophy 2006 ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own and to the best of my knowledge contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement has been made to the research of others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in this thesis. 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THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Swanson First name: Rebecca Other name/s: Lyn Abbreviation for degree as given in the University calendar: PhD School: School of Biological, Earth and Environmental Science Faculty: Science Title: A naturally occurring settlement cue for larvae of the Australian sea urchin Holopneustes purpurascens Abstract 350 words maximum: (PLEASE TYPE) The importance of chemical cues in triggering the settlement of marine invertebrate larvae has long been recognised but very few such cues have been definitively identified. Larvae of the Australian sea urchin Holopneustes purpurascens, which lives enmeshed in the fronds of macroalgae, are induced to settle by a water-soluble cue produced by the host alga Delisea pulchra. This cue was previously identified as a floridoside-isethionic acid complex. I present evidence in this thesis which supports histamine as the true settlement cue for larval H. purpurascens. The settlement cue was isolated from the polar extract of D. pulchra by bioassay-guided cation-exchange chromatography and identified as histamine using nuclear magnetic resonance spectroscopy. Algal derived and synthetic histamine at ~5 μM induced rapid settlement in 80–100 % of larval H. purpurascens. In the first study of its kind for any marine invertebrate, variation in the distribution of new recruits was compared with quantitative variation in the distribution of histamine in the habitat. More than 90 % of new recruits were found on either the foliose red alga D. pulchra or on coralline turf algae. These algae induced >90 % settlement of larvae in laboratory assays after 24 h. D. pulchra contained far higher levels of histamine than all other algae, however, the coralline algae lacked measurable histamine. Seawater collected in situ adjacent to D. pulchra induced up to 16 % settlement of older larvae and contained the highest concentration of histamine (~5 nM). With the exception of coralline algae, variation in settlement and recruitment was consistent with the variation among species histamine contents. Initial results supported a biofilm derived settlement cue from coralline algae. I also showed that older H. purpurascens larvae settle in response to lower concentrations of histamine than younger larvae and required less exposure to histamine (10 μM) in order to initiate irreversible metamorphosis. Histamine induced settlement of two other echinoids with non-feeding larvae. Histamine did not induce settlement of feeding larvae of two echinoids or settlement of non-feeding larvae of asteroids. Histamine may be a general settlement cue for echinoids with direct development. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). …………………………………………… …………………………………….. ……….……………………...… Signature Witness Date FOR OFFICE USE ONLY Date of completion of requirements for Award: i Acknowledgements I thank my supervisor Professor Peter Steinberg for his wise supervision over the years and for giving me the opportunity to work with such a wonderful group of people. I thank my Co-Supervisor Professor Rocky de Nys for his enthusiasm for science and life in general which is inspiring, and for his encouraging words particularly in the early years of this project (when confusion and doubt were supreme) and in the final weeks of writing this thesis. I would like to give special thanks to Jane Williamson for laying the groundwork for this project which I have thoroughly enjoyed researching. You have each helped me to discover a true interest in marine chemical ecology, in particular, in marine invertebrate larvae which are truly amazing creatures (not to mention very, very cute)! I should also thank my Yr 11 Biology teacher, Justine Waters, who first inspired my love of Biology. I have been very lucky to share my research experience with many fellow students and research staff in the School of Biological (Earth and Environmental) Science and the Centre for Marine Biofouling and Bio-Innovation. I sincerely hope that the friendships made during this time will last a lifetime. Fellow students, especially Megan Huggett, Sharon Longford, Nicholas Paul, Niina Tujula, Mike Taylor, Tim Charlton, Jacinta Green, Keyne Monro, Adriana Verges and Alex Campbell, have made my PhD experience all the more special. Research staff past and present; Odette Ison, Sophia McCloy, Neda Shakibaee, Louise McKenzie, Kirsty Collard, Peter Schupp, Dustin Marshall, Paul Gribben and Lachlan Yee, have been great fun to work with. I apologise for being bossy and grumpy at times, and for writing all those notes, but someone had to try and organise you lot!! Special thanks go to Tim Charlton for always happily providing advice and direction on GC-MS (the bane of this project!). Special thanks to Professor Maria Byrne, Paula Cisternas and Tom Prowse of the Evolutionary Development Laboratory at the University of Sydney for providing other larval species for histamine tests and for sharing your facilities. I thank Maria for providing helpful comments on Chapter Five and for her enthusiasm for larval biology which is inspiring. Big thanks to Jacinta for ii helping me format and ‘master’ this thesis. This was stressful enough with your expert help. I can imagine how awful the last few days would have been without you! My family have always provided great support for my academic endeavours, both emotional and financial, from the senior years of high school through to this everlasting PhD. Special thanks to my beautiful mum, Marlene, for many years of love, support and encouragement, for minding Nicholas, and for all those delicious home cooked meals which have sustained me, even many years after moving out of home!! Thanks to my dad John, and my sisters Jacqui and Karen, for their continued love and support. Thanks Aunty ‘Goo’ for your support, especially for your cooking in the last few months. Special thanks to my husband Neil for the emotional and moral support over the years. Neil has lived my PhD experience and endured the lows of such a protracted affair (I hope I am not so grumpy now this is over)! I also thank Neil’s family who have all been very interested in my work. Nicholas, my gorgeous little boy, has brought me great love and happiness. Although the arrival of Nicholas put the PhD on hold, he also put my life into perspective which helped me to finish writing this thesis in the right frame of mind. To Baby Swanson on the way, now I can think about you!! “There is one more thing…………….it’s