Marine symbiotic associations involving corals, acoel worms and their dinoflagellate algae: Initiation of symbiosis, diversity of symbionts, specificity and mode of symbiont acquisition Thesis submitted for the degree “Doctor of Philosophy” By Orit Barneah Submitted to the Senate of Tel-Aviv University August, 2005 Marine symbiotic associations involving corals, acoel worms and their dinoflagellate algae: Initiation of symbiosis, diversity of symbionts, specificity and mode of symbiont acquisition Thesis submitted for the degree “Doctor of Philosophy” By Orit Barneah Submitted to the Senate of Tel-Aviv University August, 2005 This work was carried out under the supervision of Prof. Yehuda Benayahu and Prof. Virginia M. Weis This work is dedicated to my grandmother Elsbeth Bernheim Acknowledgments I would like to express my gratitude to many people who helped me during this research. To Hudi Benayahu who guided me in the magnificent world of coral reefs. Thank you for the enthusiastic supervision, encouragement and support all along the way. To Virginia Weis who securely led me in the mysterious world of molecular biology. Thank you for the warm hospitality in Oregon State University, for the professional help and dedicated supervision. Thanks are due also to all Virginia’s lab members who helped me tremendously. To my committee members: The late Boaz Moav for his valuable advice and ideas, Ofer Mokady for his professional help and willingness to assist at any time and Anton Post for valuable assistance and advice. To Itzik Brickner for valuable assistance in the field, with histology, with ideas, with questions and most importantly for being my friend. To Lev Fishelson for his valuable assistance and willingness to share his enormous knowledge. To my colleagues abroad: To Todd LaJeunesse for teaching me the DGGE technique and assisting with data analysis and to Matthew Hooge for introducing me to the fascinating world of acoel worms and for his valuable assistance in this field. To Revital Ben-David Zaslow for valuable assistance in many realms. I owe you a lot… To Yossi Loya and all his lab members for the cooperation and assistance. To Esti Winter for assistance with histology, electron microscopy interpretation and for the readiness to help with any topic. To Amikam Shoob for the amazing photographs. To Naomi Paz for editorial assistance. To Moshe Aleksandroni for his help with photography and prints. To Varda Wexler for precious assistance and endless patience with numerous graphic projects. To Yakov Delaria for his valuable work in the Electron Microscopy. To Tali Yacobovitch, Dafna Zeevi, Noa Shenkar and Noga Sokolover for their friendship and assistance. To my lab mates who helped me above and underwater: Nissim Sharon, Shimrit Perkol- Finkel, Ettie Sapir, Sharon Levi, Mati Halperin, Anat Maoz, Idit Adler, Yoni Sharon, Ido Sela, Dror Zurel, Inbal Ginsburg and Yael Zeldman. To the undergraduate students who helped me at different stages of my work: Yaniv Nevo, Nadav Gofer, Rachel Noiman and Michal Ironi. To the staff of the Interuniversity Institute of Marine Biology at Eilat (IUI) for the kind hospitality and assistance. To all my colleagues and friends at the Department of Zoology. To my parents Uri and Esther and my sisters Ilanit and Ayelet for accompanying and supporting me along this long way ♥ Table of Contents Page List of plates and tables English Abstract 1. General Introduction 1 1.1 Research Goals 1 1.2 Background 4 1.3 Initiation of a symbiotic relationship between corals and their 6 symbiotic algae: in search of symbiosis-specific proteins. 1.4 Specificity in coral-algal symbiosis 7 1.5 Modes of symbiont acquisition by cnidarian hosts 8 1.6 Specificity in symbiotic systems and the relation to the mode of 8 symbiont acquisition 1.7 Coral hosts and algal symbionts – Molecular tools in aid for 9 classical taxonomical tools. 1.8 Molecular tools - From Restriction Fragment Length polymorphism 11 (RFLP) to Denaturing Gradient Gel Electrophoresis (DGGE) 1.9 Symbiosis involving acoelomorph worms and unicellular 12 phototrophic dinoflagellates 2. Section II. Comparative proteomics of symbiotic and aposymbiotic 14 aposymbiotic juvenile soft corals 2.1 Introduction 14 2.2 Materials and Methods 16 2.2.1 Maintenance of animals 16 2.2.2 Protein extraction from animal tissue 17 2.2.3 2D-PAGE 17 2.2.4 Analysis of 2D gels 18 2.2.5 Control for possible algal protein contamination in host gels 18 2.3 Results 18 2.4 Discussion 22 3. Section III. Diversity of dinoflagellate symbionts in Red Sea soft corals: 25 mode of symbiont acquisition matters 3.1 Introduction 25 3.2 Materials and Methods 27 3.2.1 Collection and identification 27 3.2.2 Extraction of DNA 27 3.2.3 PCR amplification and (RFLP) analysis: 28 3.2.4 DNA sequencing 28 3.2.5 Phylogenetic analysis 29 3.3 Results 30 3.4 Discussion 34 4. Section IV. Interactions involving acoelomorph worms, corals and 39 unicellular algal symbionts in Eilat (Red Sea) 4.1 Introduction 39 4.2 Materials and Methods 42 4.2.1 Field observations and collection of animals 42 4.2.2 Scanning electron microscopy of worms 42 4.2.3 Scanning electron microscopy of coral surface 43 4.2.4 Transmission electron microscopy of worms 43 4.2.5 Genetic analysis of Symbiodinium spp. symbionts from 43 corals and resident worms 4.2.6 Extractions of zooxanthellae DNA 43 4.2.7 Denaturing-gradient gel electrophoresis (DGGE) 44 4.3 Results 44 4.4 Discussion 52 5. Section V. Sexual and asexual reproduction and mode of symbiont 59 acquisition in Waminoa brickneri 5.1 Introduction 59 5.2 Materials and Methods 60 5.2.1 Collection and maintenance of animals 60 5.2.2 Histology 61 5.2.3 Transmission electron microscopy of worms 61 5.2.4 Asexual reproduction of worms 61 5.3 Results 62 5.4 Discussion 67 6. Section VI. Summary and Conclusions 71 Literature Cited 83 Hebrew Abstract I List of figures and tables Page Figure 2.1 Silver-stained 2-D gels of soluble proteins from planulae (A) and an 20 adult colony (B) of Heteroxenia fuscescens Figure 2.2 Silver-stained 2-D gels of soluble proteins from primary polyps of 21 Heteroxenia fuscescens, differing in their ages and symbiotic states Figure 2.3 Summary of changes in spot intensity through host ontogeny. 22 Enlargement of the spots marked on Figures 2.1 and 2.2. Figure 3.1 Taq I RFLP analysis of ss-RNA encoding DNA from 33 zooxanthellae of different Red Sea soft corals. Figure 3.2 Phylogenetic reconstruction of Symbiodinium spp. from 34 different soft coral host species using partial sequences of 18s rRNA gene. Figure 4.1 Stony corals inhabited by Waminoa sp. worms. 47 Figure 4.2 Scanning electron microscope images of worms isolated from Red 47 Sea stony corals. Figure 4.3 Scanning electron micrograph of a fractured acoelomorph worm. 48 Figure 4.4 Transmission electron micrographs of algal symbionts within 49 Waminoa sp. Figure 4.5 Scanning electron micrographs of the surface area of the soft coral 51 Stereonephthya cundabiluensis. Figure 4.6 Representative PCR-DGGE ITS2 fingerprints (profiles) of 51 Symbiodinium spp. Symbionts observed in coral hosts (Sty2=Stylophora pistillata, Ac6=Acropora hemprichi, PL7=Plesiastrea laxa, Tu4=Turbinaria sp. And Str2=Stereonephthya cundabiluensis) and their resident worms (w). Figure 4.7 PCR-DGGE ITS2 fingerprints of endosymbionts obtained from six 52 colonies of the stony coral Turbinaria sp. (Tu2, Tu3, Tu4, Tu5, Tu10, Tu11) And the respective profiles of the endosymbionts obtained from the worms found on each of them Figure 5.1 (A) The stony coral Plesiastrea laxa with Waminoa brickneri 64 worms. (B-F) Stages of sexual reproduction in W. brickneri. Figure 5.2 Waminoa brickneri TEM micrographs of gonads: 18 days prior to 65 egg laying. Figure 5.3 Waminoa brickneri TEM micrographs of: embryo within egg 66 capsule. Figure 5.4 Waminoa brickneri Asexual reproduction 66 Table 2.1 Tallied results from 2D PAGE of soluble proteins from 19 Heteroxenia fuscescens. Table 3.1 List of soft corals examined, their modes of reproduction and 32 developmental stage during which symbionts are acquired. Table 4.1 Occurrence of acoelomorph worms on coral hosts in Eilat (Red 46 Sea). Infestation ratio is given as number of infected colonies vs. number of colonies counted. ABSTRACT The persistence of coral reefs for millions of years in nutrient-poor waters, where sunlight can penetrate to depths occasionally exceeding 100 m, is without doubt related to the presence of numerous unicellular algal cells residing within the coral tissues. Cnidarian- algal symbiosis is considered among the most significant marine mutualisms, forming the trophic and structural foundations of coral reef ecosystems. Symbiotic systems, being based on the relationship of two different entities, have long been subjected to studies of the degree of specificity between the host and its symbionts. In regard to cnidarian-algal symbiosis, this subject represents a complex case study, which can be attributed to: (1) the taxonomy of the symbionts being still under investigation; (2) the dual mechanism of symbiont acquisition (source of symbionts varies with host taxon); and (3) the fact that there are hundreds of host species and unknown numbers of symbiont species. Various biotic and abiotic aspects have been examined in relation to specificity and many of them have been extensively dealt with. An important factor to consider in studies of host- symbiont specificity is the mode of symbiont acquisition by the host. The onset of a symbiotic relationship differs among associations. Symbionts can be vertically transmitted from host parent to offspring, or they can be acquired horizontally from the surrounding environment with each new host generation.