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 16 2.2.2 Protein extraction from 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. Direct transmission of algae from parent to offspring is uncommon among marine symbiotic systems and has only been described among members of the phylum Porifera and in a few members of the diploblastic phylum . Such acquisition takes place by way of incorporation of symbionts into the oocyte. While cnidarian-algal symbioses exhibit both strategies,

I symbiotic worms belonging to the recently established triploblastic phylum

Acoelomorpha are so far known to employ only horizontal transmission.

The initiation of a symbiotic relationship is often accompanied by morphological, physiological, biochemical and molecular changes in both partners. While in certain symbioses, such as plant-microbe endosymbioses, this biochemical and molecular interplay between the partners has been extensively studied, in others, such as cnidarian- algal symbioses, these interactions remain largely undescribed. Although numerous recent studies have focused on the breakdown of the symbiotic association, namely bleaching, understanding of the initiation of the symbiotic relationship between corals and symbiotic algae from a molecular point of view is still at an early stage.

Cnidarian-algal symbiosis is currently being extensively studied and continues to draw most research interest, especially in light of the frequent coral bleaching episodes occurring worldwide. Contrary to the well-studied cnidarians, symbiotic species belonging to the phyla Platyhelminthes and particularly Acoelomorpha, are the most understudied group of associations considering the numerical abundance, diversity of taxa, range of habitats, and geographic distribution. Recent molecular and morphological evidence suggests that members of Acoelomorpha are the most basal known triploblastic bilateria. This phylum includes members of the class Acoela, which are occasionally epizoic on corals and contain symbiotic unicellular algae. Acoela is a morphologically diverse group of small (~0.5-10 mm long) and soft-bodied worms, lacking a gut cavity and protonephridia. The occurrence of algal symbionts in both corals and worms and the

II close physical encounter of the worms with their coral hosts, intrigued me into conducting a molecular comparison of the algal symbionts in the worms vs. their coral hosts. This study shed light on the nature of a triadic symbiotic system, involving corals, worms and dinoflagellate algae.

The research of symbiotic systems is complex and involves aspects of diversity, morphology, physiology, recognition and specificity of the interacting partners, often belonging to markedly different taxa. The present research was conducted in Eilat

(northern Red Sea), involved various aspects of symbiosis and dealt with questions regarding the coral host, the algal symbionts and, finally, a glimpse into a whole new symbiotic relationship linking corals, acoel worms and symbiotic algae.

In the first part of the study I searched for symbiosis-related proteins in symbiotic vs.

aposymbiotic primary polyps of the Red Sea soft coral Heteroxenia fuscescens, using two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE). The second part of the study concentrated on the clade identity of algal symbionts in Eilat’s soft coral hosts in relation to the mode of symbiont acquisition, using RFLP (Restriction Fragment

Length Polymorphism) of the algal 18S rRNA gene. Finally, in the third and fourth parts of this work I examined the symbiotic relationship between corals and epizoic acoel worms, belonging to the genus Waminoa, as well as the sexual reproduction and algal acquisition mode of the newly discovered species Waminoa brickneri.

III In choosing a system to investigate the initial stages of coral-algal symbiosis, the best

strategy is to study an association with horizontal transmission, in which there are both

aposymbiotic and symbiotic early host ontogenetic stages. In the present study I used 2-

dimensional polyacrylamide gel electrophoresis to compare patterns of proteins synthesized in symbiotic and aposymbiotic primary polyps of the Red Sea soft coral

Heteroxenia fuscescens. This is the first work to search for symbiosis-specific proteins during the natural onset of symbiosis in early host ontogeny. The protein profiles reveal changes in the host soft coral proteome through development, but surprisingly virtually no changes in the host proteome as a function of symbiotic state. There are several suggested explanations for the uniformity of proteome patterns between symbiotic and aposymbiotic hosts. It is possible that changes do occurr but are not being detected. This could be due to a variety of factors, including slow protein turnover with age of the host proteome, transient expression of symbiosis-specific or enhanced proteins, and expression of these proteins in very low quantities below detection levels. If symbiosis- specific protein expression is indeed limited to those cells housing the symbionts, then these host cells number only in the hundreds to thousands in the early weeks of the symbiosis. Against a background of many thousands of cells comprising a juvenile polyp, any symbiosis-specific protein signal might be lost. An example that further demonstrates this possibility is found in Euprymna scolopes, where a differential result was obtained only for those symbiosis-specific organs that had been dissected away from the rest of the animal. Since in Cnidaria algal symbionts are not housed in such a specific

location, detecting these symbiosis-specific changes is hindered by the high non-

symbiosis cell background. In conclusion, the onset of symbiosis between H. fuscescens

IV and Symbiodinium sp. is not accompanied by any gross changes in the host proteome in the first weeks of the association.

I investigated the clade identity of symbionts in soft coral hosts (Eilat, Red Sea) in

relation to their hosts’ mode of symbiont acquisition and found for the first time that all

hosts using horizontal transmission harbored symbionts belonging to Clade C while those

with vertical transmission uniquely harbored symbionts from Clade A. There is evidence from studies performed in the Caribbean that Clade A zooxanthellae are shallow-water specialists and are relatively stress tolerant or ‘weedy’ (Rowan 1998). It has also been shown that Clade A zooxanthellae are widely tolerant to temperature change and are the only clade capable of synthesizing mycosporine-like amino acids (MAAs). Together, these studies suggest that Clade A algae may be well adapted to cope with Eilat’s environmental conditions. We suggest that Clade A symbionts, capable of coping with a wide array of environmental conditions, evolved as optimal vertically transmitted symbionts that did not fail their host and thus persisted. The limitation of Clade A symbionts to hosts with vertical transmission suggests a coevolution of hosts and symbionts. Clade C symbionts, characterized by large sub-clade variability, are found in corals with horizontal transmission and, most probably, each of their genotypes exhibits a more specialized set of physiological capabilities. Based on my results from soft corals and on the recent literature on stony corals (Montipora and Porites respectively), I hypothesized that corals which vertically transmit their symbionts will tend to have genetically distinct symbionts, which differ from those found in corals with horizontal transmission. In this study, the distinctive feature is symbiont clade,

V

“Spotted” coral colonies in Eilat (Red Sea) were noted already more than a decade ago,

yet, this phenomenon has only recently been attributed to infestations of acoel worms of

the genus Waminoa. A recent systematic revision of Waminoa from Eilat revealed a new species, W. brickneri. Fourteen coral species were found to be infested by acoel worms at a depth range of 2 to 50 meters. The host species were all zooxanthellated and included both massive and branching stony corals and a soft coral. Worms from all hosts were identified as belonging to the genus Waminoa and contained two distinct algal symbionts differing in size. The smaller one was identified as Symbiodinium sp. and the

larger one is presumed to belong to the genus Amphidinium. Comparison between worm-

infested and non-infested branches of a soft coral, shows that the former have distinct

surface microvilli while the latter are covered by mucus. In most situations, the

Symbiodinium spp. residing in the host corals were genetically different to those present

within the epizoic worms, hence predation on coral tissue by the worms is not likely to

have occurred. The subsequent monitoring of sexual reproduction and the discovery of

the mode of algal acquisition in the acoel W. brickneri supported the molecular results

obtained. Sexually mature worms were removed from the stony coral Plesiastrea laxa

and raised in the laboratory. Eggs were detected 18 days after collection of the worms

and hatched 4 days later. Histological sections performed on sexually mature worms

showed an ovary with oocytes containing two distinct algal endosymbionts within their

ooplasm. The discovery of maternal transmission of two distinct types of algal symbionts

W. brickneri is the first documentation of this mode in any triploblastic organism studied

to date. This finding suggests that these worms can maintain their algal symbionts from

VI generation to generation without relying on acquisition from external sources, such as

their coral host, for supply of symbionts.

This study touches upon different aspects of symbiotic systems and mainly focuses on

specificity. Through the investigation of genetic identity of algal symbionts a connection

has been shown between the mode of symbiont acquisition in soft corals hosts at Eilat

and the genotype of their symbionts, suggesting a coevolution of both partners.

Furthermore, the use of similar molecular tool demonstrate that algal symbionts in

several coral hosts differ from those found in their epizoic acoel worms, indicating that

worms should most probably not rely on predation of coral tissues in order to obtain their

symbionts. Further study conducted on the sexual reproduction of these worms clarified

the picture as it was found that the worms vertically transmit their symbionts at the

oocyte stage, hence showing that they maintain their symbionts from generation to

generation. Vertical transmission of algal symbionts as shown in Waminoa brickneri is

the first evidence for this mode in any triploblastic organism studied to date. The search

for symbiosis-related proteins originating in the coral hosts in the initiation of the

symbiotic relationship, revealed no marked differences between protein profiles obtained

from aposymbiotic and symbiotic primary polyps of the soft coral Heteroxenia

fuscescens.

The results of this study encourage a thorough globally-based investigation of corals that

employ vertical transmission of algal symbionts in order to gain a broad understanding of

the mechanisms which underlie the persistence of such corals in a changing environment.

VII Moreover, the uniformity of protein profiles of aposymbiotic and symbiotic primary polyps suggests that future studies regarding the fascinating stage of symbiosis initiation should employ a different approach, preferably involving a finer resolution tool. Finally, the disclosure of the triple symbiosis system involving corals, worms and unicellular algae as discovered in Eilat, will undoubtedly focus future attention on the specificity, physiology and diversity of the interacting parties.

VIII SECTION I

GENERAL INTRODUCTION

1.1 Research Goals

The present research focuses on symbiotic interactions involving coral hosts and dinoflagellate algae belonging to the genus Symbiodinium as well as Acoelomorph worms, harboring Symbiodinium and an additional dinoflagellate alga, found epizoic on living corals. This work includes themes which center on the host, the symbionts as well as the interaction between the partners. The first objective of this research was to look into changes in the coral host proteome during the initiation of the symbiosis with zooxanthellae. The results of this part should have led me subsequently to investigate certain proteins and genes which are involved in the commencement of coral-algal symbiosis. As the research progressed and results were in hand, certain adaptations in the original plan were needed. One of the basic questions that were asked in the beginning of my research was what clade of zooxanthellae is found in the coral Heteroxenia fuscescens. This coral, was extensively studied (Achituv & Benayahu, 1990; Ben-David-

Zaslow et al., 1999; Yacobovitch et al., 2003) and its mode of reproduction and aspects of symbiont acquisition were well documented. Moreover, a protocol for controlled infection of primary polyps with freshely isolated zooxanthellae was established

(Yacobovitch et al., 2003). Hence, this coral was chosen as a model for the first part of the research. After identifying the clade in H. fuscescens. and since scarce data existed on the diversity of algal symbionts in other coral hosts in the Gulf of Eilat, I was intrigued to find out what clades of zooxanthellae are found in other soft coral hosts in this area especially in light of the fact that most of them were studied in terms of sexual reproduction and their mode of symbiont acquisition were described. These findings

1 enabled me to investigate the possible connection between symbionts’ identity and the

mode of their acquisition by a host. This question constitutes the second part of my research.

The third and fourth parts of my research are tied together and evolved during my field work in Eilat, after I came across coral colonies that were associated with worms. The worms were found to contain zooxanthellae and this fact thus raised a working hypothesis which was tested. The association of corals with symbiotic worms (containing algae) was rarely studied previously. Therefore, I was motivated to examine this symbiotic interaction.

• Section I of the study is entitled: Comparative proteomics of symbiotic

and aposymbiotic juveniles of the soft coral Heteroxenia fuscescens.

In this part I compared patterns of proteins synthesized in symbiotic and

aposymbiotic primary polyps of the Red Sea soft coral Heteroxenia

fuscescens in order to find symbiosis-specific proteins that might be

involved in the initiation of the symbiotic relationship between a coral

host and its symbiotic algae.

Research question: Are there symbosis-related genes/proteins which are

expressed in the symbiotic primary polyps of the soft coral H. fuscescens

at different time intervals post infection with freshely isolated algal

symbionts ?

• Section II of the study is entitled: Diversity of dinoflagellate symbionts

in Red Sea soft corals: mode of symbiont acquisition matters.

2 This part investigated the clade identity of symbionts in soft coral hosts

(Eilat, Red Sea) in relation to the mode of symbiont acquisition by the

hosts.

Research question: How does the mode of symbiont acquisition by soft

coral hosts affect their symbionts’ identity?

• Section III of the study is entitled: Three party symbiosis: acoelomorph

worms, corals and unicellular algal symbionts in Eilat (Red Sea). In

this section I report the presence of acoelomorph worms epizoic on 14

zooxanthellated coral species in the reefs of Eilat (Red Sea). The worms

all belong to the genus Waminoa; those isolated from the stony coral

Plesiastrea laxa belong to a new species, Waminoa brickneri (Ogunlana et

al., 2005). I studied the distribution of the worms on different coral hosts,

the identity and position (within the worm) of the algal symbionts and

possible effects of the presence of worms on the coral host.

Research questions:

1. Do worms and their coral hosts possess the same genotype of algal

symbionts?

2. Do the worms physically harm their coral hosts

• Section IV of the study is entitled: Sexual and asexual reproduction and

mode of symbiont acquisition in the acoelomorph Waminoa brickneri.

In this part I studied the sexual and asexual reproduction process and the

3 mode of symbiont acquisition in the newly discovered species W.

brickneri.

Research Question: What are the features of sexual and asexual reproduction in

the acoelomorph worm Waminoa brickneri and what is the mode of symbiont

acquisition by the worms?

The four chapters of my research touch upon basic themes in symbiosis: The initiation of a symbiotic relationship as reflected from a coral-algal system and a worm-algal system;

The mode of symbiont acquisition by a coral host and its relation to symbiont identity as revealed in open vs. closed systems of symbiont acquisition as well as the mode of symbiont acquisition in a new species of symbiotic acoelomorph worm; and specificity in soft coral-algal symbiosis as recorded in Eilat's coral reef.

1.2 Background

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. Symbiosis facilitates reef-building by animals by providing them with the energy-generating capacity of the plants (Veron, 2000). Endosymbiotic relationships involving members of the phylum Dinophyta (dinoflagellates) are known to occur with hosts belonging to phyletically diverse groups, including Protozoa, Porifera, Cnidaria, Turbellaria and

Mollusca (Trench, 1987; Davies, 1993; McCoy & Balzer, 2002). The most widespread dinoflagellate symbioses are those with members of the phylum Cnidaria, particularly with the Class (Davies, 1993). The animal host gives the algae a stable medium in which to live and one which is exposed to sunlight (Veron, 2000). The physiological advantages attributed to the algae in this relationship are diverse and include: the 4 enhancement of calcification (Goreau, 1961; Barnes & Chalker, 1990), translocation of

metabolites from symbiont to host (Davies, 1993; Porter, 1974), removal of metabolic

wastes (Yonge and Nicholls, 1931; Goreau, 1961) and concentration and recycling of the

limited nutrients, including nitrogen and phosphate (eg., Muscatine & Porter, 1977). This

symbiotic relationship has led to the creation of the largest and most spectacular

structures made by any living organisms: namely, coral reefs (Veron, 2000). An exact

dating of the formation of the symbiotic association between algae and invertebrates is

not known, although it is assumed that the association of scleractinian corals and

dinoflagellates was initiated in the mid Triassic, around 230 million years ago (Trench,

1993).

"Zooxanthellae" is a general descriptive term for dinoflagellate symbiotic algae that live

in animals (Muller-Parker & D'Elia, 1997). Currently, eight genera in four (or five)

classical orders of dinoflagellates are recognized as endosymbionts in marine

invertebrates and protists. The most studied genus in this paraphyletic group is

Symbiodinium (see: Baker, 2003). The dinoflagellates comprise a diverse group of mostly

free-swimming single-celled microscopic planktonic algae that exhibit a variety of

feeding modes ranging from photoautotrophy to heterotrophy. Zooxanthellae are able to

photosynthesize and contain characteristic dinoflagellate pigments (diadinoxanthin,

peridinin) in addition to chlorophylls a and c. They are brown or yellow-brown in color

(Muller-Parker & D'Elia, 1997). Zooxanthellae can live independently of their animal host, and while at this stage, they possess two flagella, which enable their motility. Inside their animal host, zooxanthellae are usually found in the coccoid stage, lacking flagella and thus nonmotile (Muller-Parker & D'Elia, 1997).

5 1.3 Initiation of a symbiotic relationship between corals and their symbiotic algae: in search of symbiosis-specific coral proteins.

The initiation of a symbiotic relationship is often accompanied by morphological, physiological, biochemical and molecular changes in both partners (Taylor, 1973;

Douglas, 1994; McFall-Ngai & Ruby, 1991; Montgomery & McFall-Ngai, 1994). While in certain symbioses, such as plant-microbe endosymbioses, this biochemical and molecular interplay between the partners has been extensively studied (e.g., Bestel-Corre et al., 2004), in others, such as cnidarian-algal symbioses, these interactions remain largely undescribed. Although numerous recent studies have focused on the breakdown of the symbiotic association, namely bleaching (e.g., Brown, 1997; Lesser, 1997; Hoegh-

Guldberg, 1999; Toller et al., 2001; Diaz-Pulido & McCook, 2002; Brown et al., 2002;

Franklin et al., 2004), examination of the initiation of the symbiotic relationship between corals and symbiotic algae from a molecular point of view is still at an early stage (Weis

& Levine 1996; Kuo et al., 2004). In choosing a system to investigate the initial stages of coral-algal symbiosis, the best strategy is to study an association with horizontal symbiont transmission, in which there are both aposymbiotic (symbiont-free) and symbiotic early host ontogenetic stages. To date, studies that have examined symbiosis- specific genes and proteins of cnidarians have focused on adult sea anemones (Weis &

Levine, 1996; Weis & Reynolds 1999; Reynolds et al 2000; Chen et al., 2004; Kuo et al.,

2004).

To discover possible symbiosis-related proteins, the present study compared patterns of proteins synthesized in symbiotic and in aposymbiotic primary polyps of the Red Sea soft coral Heteroxenia fuscescens. I used 2D PAGE and silver stain, which are considered the best approach in the biotechnology industry for the detection of minor

6 proteins (Nishihara & Champion, 2002). O’Farrell (1975) first introduced two-

dimensional gel electrophoresis for the separation of proteins; today it serves as the core

technology of proteomics. This technique allows the simultaneous resolution of

thousands of proteins in one separation procedure (Görg, 1998). The separation is made

according to two independent properties in two discrete steps: the first-dimension step,

isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI); the

second-dimension step, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), separates

proteins according to their molecular weights (MW) (Berkelman & Stenstedt, 1998).

1.4 Specificity in coral-algal symbiosis

Symbiotic systems, being based on the relationship of two different entities, have always been subject to a study of the degree of specificity between the host and symbionts (see

Douglas, 1994).

It is well known today that specificity in cnidarian-algal symbiosis should be viewed in

two directions: host specificity (the specificity of hosts for a particular range of symbionts) and symbiont specificity (the specificity of symbionts for a particular range of

hosts) (Baker, 2003). More than a decade of intensive research around the world has demonstrated that there are generalist hosts species and symbiont types while others tend to be very specialists (Baker, 2003)

The complexity in studying specificity in cnidarian-algal symbiosis can be attributed to:

(1) the taxonomy of the symbionts is under investigation; (2) there are two mechanisms

of symbiont acquisition; and (3) there are hundreds of host species and unknown numbers

of symbiont species. There are various biotic and abiotic aspects that have been examined

in relation to specificity and many of them have been dealt with (see Baker, 2003). These

include possible effects of depth on the presence of symbionts (Rowan & Knowlton,

7 1995), biogeography and its relation to identity of symbionts in certain coral hosts

(Rodriguez-Lanetty et al., 2003), and ontogeny of the coral host and its relation to

symbionts identity (do juveniles and adults harbor the same symbionts) (Coffroth et al.,

2001). An important factor to consider in studies of host-symbiont specificity is the

mode of symbiont acquisition by the host (Rowan, 1998). The co-occurance of coral host

species which acquire their symbionts anew with each life cycle side by side with hosts

that transfer their symbionts faithfully from generation to generation is intriguing in terms

of the identity of the algal symbionts found in such hosts. While in several symbiotic

systems, such as aphid-bacteria and squid-bacteria (one species of squid with one species

of bacteria), this issue has been resolved (Douglas, 1994), in others, including Cnidarian-

algal symbiosis, data is still missing.

1.5 Modes of symbiont acquisition by cnidarian hosts

The mechanisms of acquisition of dinoflagellate symbionts by the sexually-derived

progeny of invertebrate hosts can be divided into two basic categories: (1) acquisition by

maternal inheritance (a closed system) also known as vertical transmission, and (2)

acquisition from the environment by either larval or adult stages (an open system) also

known as horizontal transmission (Trench, 1987; Douglas, 1995). In the latter case, hosts

can generally form associations with a broad range of potential symbionts present in the

water column, while in the former, symbionts are transmitted faithfully from parent to

offspring and are virtually “locked” within their hosts (Douglas, 1998).

1.6 Specificity in symbiotic systems and the relation to the mode of symbiont acquisition

8 Vertical transmission of symbionts tends to restrict the distribution of those among hosts.

If this mode of transmission is sustained through many host generations and horizontal

transmission is entirely absent, the symbionts will be limited to a single host lineage, and

the phylogeny of the symbionts will come to mirror that of the host (Douglas, 1995). As

cnidarian hosts are characterized by duality of symbiont acquisition modes (different

species of corals apply different modes of symbiont acquisition) the specificity issue is

far more complex. The phylogeny of Symbiodinium, as determined from 18S rRNA gene

sequence, shows no concordance with the phylogeny of its cnidarian hosts (Rowan &

Powers, 1991a). Congruence between the cnidarian and Symbiodinium lineages is probably precluded by the evolutionary plasticity of reproductive traits, including symbiont transmission, in the cnidarian hosts (Fautin, 1991).

1.7 Coral hosts and algal symbionts - Molecular tools in aid for classical taxonomical tools

Taxonomic studies of invertebrate hosts and symbiotic algae are in the process of gaining a new dimension based on molecular analysis. The era of rapidly evolving molecular

techniques has opened up new opportunities in the study of systematics. One great

challenge confronting researchers dealing with molecular systematics is that of how to

link the data obtained by traditional morphological-based techniques with the newly acquired molecular data, in order to bridge possible gaps between the two types of methodologies and create a reliable picture. Veron (1995) has commented that in the long-term, molecular techniques may displace morphology as the primary basis for differentiating species. Looking into the systematic world of both corals and their symbiotic dinoflagellates reveals two markedly different case studies: stony corals,

9 characterized by diverse skeletal morphology have been described and studied since the very early 18th century (Veron, 2000). The development of SCUBA contributed

enormously to the study of coral reef fauna in general and stony corals in particular, and

consequently a great deal is known about corals today. Most coral species likely to be

encountered have already been described (Veron, 2000). In contrast, classical taxonomic

studies of their zooxanthellae have been hindered by an apparently uniform algal

morphology. Moreover, the lack of observed sexual reproduction in this group precludes

the use of the biological species concept in order to define species boundaries (Baker

2003). Another obstacle encountered by researchers documenting diversity in

Symbiodinium, is the difficulty of growing these microalgae in culture for morphological description (Rowan, 1998; Baker, 2003). Thus, traditional taxonomic tools have failed to estimate the correct magnitude of diversity of zooxanthellae.

The research of symbiotic systems is complex and involves aspects of diversity, morphology, physiology, recognition and specificity of both partners, often belonging to markedly different taxa. Douglas (1994) considered that a firm grasp of the taxonomy of the interacting organisms in symbiosis is a prerequisite for any investigation of specificity. The study of coral-algal specificity seemed straightforward in the 1960s, when Symbiodinium microadriaticum was declared to be the universal symbiont in all marine cnidarians hosting unicellular algae (Freudenthal, 1962), but this concept did not last for long. Early studies dealing with isoenzyme and soluble-protein patterns in algae cultured from 17 host species revealed differences, which were interpreted as resulting from genetic variability (Schoenberg & Trench, 1980a). The advent of molecular tools has resulted in the birth of the field of zooxanthellae molecular phylogenetics and systematics. Zooxanthellae were found to be taxonomically highly diverse and were 10 divided initially into five distinct clades, A, B, C, D and E, based on ribosomal RNA

gene sequences and RFLP patterns (Rowan & Powers 1991a; Rowan & Powers 1991b;

Rowan, 1998; LaJeunesse, 2001; Toller et al., 2001). These clades represent subgeneric taxonomic levels, and each contains one or more taxa, as revealed by denaturing gradient gel electrophoresis of the internal transcribed spacer 2 region (LaJeunesse, 2001, 2005;

LaJeunesse et al., 2004).

1.8 Molecular Tools - From Restriction Fragment Length Polymorphism (RFLP) to Denaturing Gradient Gel Electrophoresis (DGGE) The pioneering work of Rowan & Powers (1991,a,b) on molecular phylogeny of

Symbiodinium spp. using RFLP of the DNA gene encoding the ssRNA inspired researchers to continue their efforts to resolve Symbiodinium diversity. This has led to the publication of many studies that have expanded our knowledge of the identity of algal symbionts in a growing number of invertebrate hosts, corals in particular, in relation to various ecological variants such as biogeographical location, depth, illumination etc. (see

Baker, 2003). Despite the wide use of the RFLP method on the gene encoding the ssRNA, it is common knowledge that the gene is too conserved and not suitable for species level distinctions (McNally et al., 1994; Baker & Rowan, 1997). Consequently, other regions of the nuclear genes encoding ribosomal RNA, such as partial large subunit

(LSU) and internal transcribed spacers (ITS1 and 2) and 5.8S have also been analyzed

(see Baker, 2003). These studies have recognized seven distinct clades (A to G) of

Symbiodinium, which are robust despite differences in the methodology used (Baker,

2003).

A comprehensive study of Symbiodinium diversity around the world, using the internal transcribed spacer (ITS) regions, was initiated by LaJeunesse (2001). The ITS region 11 consists of ITS1, 5.8S and ITS2, and typically provides phylogenetic resolution at or below the species level (LaJeunesse, 2001). LaJeunesse implemented the use of

Denaturing Gradient Gel Electrophoresis (DGGE), which offers a powerful tool for the detection of single base changes in DNA, to the elucidation of Symbiodinium diversity.

The combined use of the DGGE technique and DNA sequence analysis resulted in both visual and molecular capabilities of detecting changes in genotype (LaJeunesse, 2001,

2002; LaJeunesse et al., 2003, 2004). This technique has paved the way for obtaining an improved phylogenetic resolution of Symbiodinium, enabling the use of sub-clades instead of clades, and thus finer scale of resolution for the study of specificity in cnidarian-algal symbiosis (LaJeunesse et al., 2003, 2004).

This study involved two aims that required molecular tools for the identification of algal symbionts: (1) Disclosing the diversity of algal symbionts in soft corals from Eilat; and

(2) Comparing algal symbionts in acoelomorph worms in relation to the algal symbionts of their coral hosts. This research first implemented the RFLP and subsequently the

DGGE technique.

1.9 Symbiosis involving acoelomorph worms and unicellular phototrophic dinoflagellates

Symbiosis involving unicellular phototrophic dinoflagellates occurs in members of the

Phylum Acoelomorpha, previously known as the Order Acoela within the Phylum

Platyhelminthes (McCoy & Balzer, 2002; Baguna & Riutort, 2004). Contrary to the well- studied cnidarians, symbiotic platyhelminth worms and particularly acoelomorphs are the most understudied group of associations considering their numerical abundance, diversity of taxa, range of habitats and geographic distribution (McCoy & Balzer, 2002). Recent

12 molecular and morphological evidence suggests that members of the phylum

Acoelomorpha are the most basal known triploblastic bilateria (Baguna & Riutort, 2004;

Ruiz-Trillo et al., 2004). This phylum includes the classes Nemertodermatida and Acoela.

The latter is a morphologically diverse group of small (~0.5-10 mm long) and soft-bodied

worms (Hooge et al., 2002), lacking a gut cavity and protonephridia (Tyler, 2003). To

date, there are over 340 known acoel species assigned to 21 families (Tyler et al., 2005).

Three of the families (Convolutidae, Sagittiferidae, and Haploposthiidae) contain species with symbiotic unicellular algae (McCoy & Balzer, 2002). The algae that may form a symbiosis with acoels belong to separate phyla, including the Bacillariophyta (diatoms),

Dinophyta (dinoflagellates) and Chlorophyta (green algae) (McCoy & Balzer, 2002).

Symbiotic marine acoels are common in littoral, sublittoral and pelagic environments and occur in temperate as well as tropical habitats (McCoy & Balzer, 2002). Symbiotic acoel worms that are epizoic on corals include Haplodiscus sp. from reefs near Micronesia

(Trench & Winsor, 1987), Waminoa litus, Waminoa sp. 1, 2 and Convolutriloba hastifera from North Queensland, Australia (Winsor, 1990).

Studies on symbiotic acoels primarily address the morphology of the worms (Winsor,

1990; Trench & Winsor, 1987). The nature of the symbiotic association i.e., the possible benefits or disadvantages of each of the three associated partners - namely worms, corals and algal symbionts, are relatively unexplored. Moreover, the source of algal symbionts in acoelomorph worms epizoic on corals and the mode of their transmission have never been studied.

13 SECTION II

Comparative proteomics of symbiotic and aposymbiotic

juvenile soft corals

2.1 Introduction

The initiation of a symbiotic relationship is often accompanied by morphological, physiological, biochemical and molecular changes in both partners (Taylor, 1973;

Douglas, 1994; McFall-Ngai & Ruby, 1991; Montgomery & McFall-Ngai, 1994). While in certain symbioses, such as plant-microbe endosymbioses, this biochemical and molecular interplay between the partners has been extensively studied (e.g. Bestel-Corre et al., 2004), in others, such as cnidarian-algal symbioses, these interactions remain largely undescribed. Although numerous recent studies have focused on the breakdown of the symbiotic association, namely bleaching (e.g. Brown, 1997; Lesser 1997; Hoegh-

Guldberg, 1999; Toller et al., 2001; Diaz-Pulido & McCook, 2002; Brown et al., 2002;

Franklin et al., 2004), examination of the initiation of the symbiotic relationship between corals and symbiotic algae from a molecular point of view is still at an early stage (Weis

& Levine 1996; Kuo et al., 2004). In choosing a system to investigate the initial stages of coral-algal symbiosis, the best strategy is to study an association with horizontal transmission, in which there are both aposymbiotic and symbiotic early host ontogenetic stages.

To date, all studies that have examined symbiosis-specific genes and expressed proteins in the host, have been on adult sea anemones. The temperate sea anemone Anthopleura elegantissima occurs naturally in both the symbiotic and aposymbiotic state and has been

14 used for the identification of symbiosis-related proteins (Weis & Levine, 1996; Weis &

Reynolds 1999; Reynolds et al 2000). Similarly, the tropical sea anemone, Aiptasia

pulchella, which can be grown in a symbiotic state, or rendered aposymbiotic following

treatment with 3-(3,4-Dichloro-Phenyl)-1,1-Dimethyl-Urea (DCMU) or prolonged

incubation in darkness, has been used recently in two studies aimed at the

characterization of symbiosis-related genes (Chen et al., 2004; Kuo et al., 2004).

In this study we compare patterns of proteins synthesized in symbiotic vs. aposymbiotic

primary polyps of the soft coral Heteroxenia fuscescens. We used 2D PAGE and silver

stain, which is considered the ‘gold standard’ in the biotechnology industry for detection

of minor proteins (Nishihara & Champion, 2002). Silver staining can result in high

sensitivity with as little as 0.5 ng of protein detected (Nishihara & Champion, 2002),

however the linear dynamic range of this technique is reported to be less than that of other protein detection techniques (Smales et al., 2003). This is the first work to search for symbiosis-specific proteins during the natural onset of the symbiosis in early host ontogeny. H. fuscescens is a common zooxanthellate soft coral occurring on the reefs of the northern Red Sea (Benayahu, 1985). It is hermaphroditic and broods planulae

(Benayahu, 1991) that are released from the parent nearly year-round (Ben-David-

Zaslow et al., 1999). All stages of planular morphogenesis, from egg to planula, occur

while the embryo is coated by the original egg mesogleal coat derived from the parent

colony. Hatching from this coat occurs as late as immediately prior to planulation

(Benayahu et al., 1989b). Planulae lack zooxanthellae upon release and algal acquisition

occurs at an early primary polyp stage (Yacobovitch et al., 2003). Populations of primary

polyps of identical age can be maintained in both the symbiotic and aposymbiotic states

15 for up to two months (Yacobovitch et al., 2003) and there are no differences in the timing and sequence of morphogenetic events during metamorphosis between symbiotic and aposymbiotic primary polyps in the laboratory (Yacobovitch et al., 2003). Therefore, H. fuscescens provides an ideal system for the study of protein and gene expression during the onset of symbiosis.

2.2 Materials and Methods

2.2.1 Maintenance of animals: Collection of planulae from Heteroxenia fuscescens was carried out in Eilat (Red Sea) following the methodology described in Yacobovitch et al.

(2003). Planulae released from each adult colony (a batch) were counted and divided into groups of 50 planulae. Each group was placed in a 50 ml plastic container with 0.45 μm

filtered sea-water (FSW). Containers were placed in an incubator (Yihdern, LE-509) set

to the temperature of ambient seawater during the same time period in Eilat. The light regime was 12 h light (30 μmol quanta m-2 s-1): 12 h dark. Half of the water in each

container was changed every other day. Planulae underwent metamorphosis after 10-20

days. Soon after metamorphosis occurred, infection with freshly isolated zooxanthellae

obtained from an adult colony (see Yacobovitch et al., 2003) was performed on one half

of the batch. Polyps were inspected under a light microscope to verify symbiotic state.

Aposymbiotic and symbiotic primary polyps were frozen in liquid nitrogen at different

time intervals after infection (3 days, 1, 2, 3, 4, 6 weeks). To process animals for

freezing, polyps were detached from the plastic containers using a glass pipette fitted

with a syringe needle, washed in 0.22 μm FSW, transferred to microfuge tubes (50

polyps per tube), placed in liquid nitrogen and stored at –80ºC prior to further analysis.

Polyp age was calculated from the day of planula release. A total of 3031 primary polyps

16 were raised, belonging to six different mother colonies. In addition, samples containing

50 planulae and 2-3 polyps from several mature coral colonies were frozen.

2.2.2 Protein extraction from animal tissue: Thawed samples were homogenized at

4ºC in a glass tissue grinder in 100 μl extraction buffer (EB: 40 mM Tris, 10 mM EDTA pH 7.4) with a protease inhibitor mix (Sigma, St. Louis, MO). The homogenate was transferred to a microfuge tube and the grinder was washed with an additional 50 μl of

EB. Extracts were centrifuged for 12 min at 12000 g at 4ºC. The supernatant fluid, containing the soluble fraction of coral proteins was removed, and protein concentration was determined spectrophotometrically using a Coomassie assay (Pierce, Rockford, IL).

It is important to note that algal cells were removed unbroken during centrifugation.

2.2.3 2D-PAGE: The Multiphor II flatbed system (Amersham, Pharmacia, Piscataway,

NJ) was used according to the manufacturer’s instructions. Immobiline Dry Strip IEG gels (non linear PH gradient of 3-10 18 cm) were used to resolve proteins in the first dimension. The strips were first soaked overnight in rehydration solution (8 M urea, 2%

Ampholytes, 0.5% Triton-X, DTT and a few grains of Bromophenol blue) containing 40

μg of protein. The first dimension was run at 20ºC for 19.5 h (0.01 h 500V, 5 h gradient to 3500V followed by 14.5 h at 3500V). IEF gels not immediately used for the second dimension were frozen at -80ºC. immediately prior to the second dimension run, IEF gels were incubated successively in 2 equilibration solutions for 10 min each. The solutions contained (I) 50 mM Tris-HCl buffer (PH=6.8) containing 6M urea, 30% glycerol, 1%

SDS and 0.8% DTT, and (II) 50 mM Tris-HCl buffer (PH=6.8) containing 6 M urea, 30% glycerol, 1% SDS and 7.2% Iodoacetamide and a few grains of bromophenol blue.

Precast gels ExcelGel® SDS 12-14% gradient, 245x180x0.5 mm were used to resolve

17 proteins in the second dimension. The second dimension was run at 15ºC for a total of 3.5 h at 1000V. Low molecular weight protein standards (Pharmacia) were run on each gel alongside each sample. Gels were silver stained (methods modified from Heukeshoven &

Dernick, 1985), covered with mylar, soaked in 1% glycerol and air-dried overnight at room temperature.

2.2.4 Analysis of 2D gels: For each developmental stage of H. fuscescens, 2-3 replicate samples were resolved on gels. Each sample was compared to the other replicates for the presence or absence of proteins. No marked differences were detected between replicates.

Gels were analyzed pairwise by eye for differences in their protein patterns by overlaying the gels on a light table.

2.2.5 Control for possible algal protein contamination in host gels: Despite our procedures to remove symbiotic algae unbroken from symbiotic host tissues, it is possible that some algae could break and that algal protein could contaminate host protein extracts. If this were true then algal proteins would mistakenly be identified as symbiosis- enhanced host proteins. To avoid this, a protein extract from freshly isolated algae was resolved using 2D PAGE and compared to all the symbiotic host protein gels.

2.3 Results

We obtained 22 protein profiles of Heteroxenia fuscescens across a range of ontogenetic stages and symbiotic states (see Table 2.1). Figures 2.1A and B show representative gels of aposymbiotic planulae and symbiotic adult colony respectively. Profiles of primary polyps contained approximately 170 spots. Figure 2.2 shows three pairs of protein profiles obtained from primary polyps at ages 3, 4 and 6 weeks. Each pair of gels shows the profiles of aposymbiotic (apo) and symbiotic (sym) polyps derived from the same

18 batch of planulae. The profiles of the symbiotic polyps represent 4 different ages of

polyps as well as 3 different times after infection with algae, 1, 2 and 4 weeks weeks respectively. Comparisons of protein profiles of aposymbiotic and. symbiotic primary polyps revealed no consistent differences regardless of polyp age or time after infection.

Overall, we found changes in protein expression that corresponded only to the developmental stage of the samples. Highlighted spots in Figs. 2.1 and 2.2 indicate examples of proteins that are differentially abundant as a function of host age. Each

differential protein is marked with a colored geometric form. Triangles surround spots that intensify with age; inverted triangles - those that decrease in expression with age; and rectangles - those that intensify only in the polyp stage (See Figs.2.1, 2.2). Spots that are unique to a certain developmental stage are marked with a circle. The circled spot in Fig.

2.1A is unique to the planula stage. This 26 kD protein is likely a yolk protein, similar to that described in larval Fungia scutaria (Schwarz et al., 1999). Changes in expression through time of the other marked spots are summarized in Figure 2.3.

Table 2.1 Tallied results from 2D PAGE of soluble proteins from Heteroxenia fuscescens.

Age of Year # days or # of samples polyps collected weeks post (weeks) infection Planulae 2000 - 1 3 2001 3 d 1 sym, 1 apo 3 2000 1 w 1 apo 3 2001 1 w 1 sym, 1 apo 4 2003 2 w 1 sym, 1 apo 5 2001 2 w 1 sym, 1 apo 5 2003 3 w 1 sym 6 2000 2 w 1 sym 6 2000 3 w 1 sym, 1 apo 6 2001, 2003 4 w 3 sym, 3 apo Adult 2003 - 2

19

Figure 2.1 Silver-stained 2-D gels of soluble proteins from planulae (A) and an adult colony (B) of Heteroxenia fuscescens. Triangles surround spots that intensify with age; inverted triangles - those that decrease in expression with age; rectangles - those that intensify only in the polyp stage and circles - those that are unique to the planulae stage. The horizontal dimension represents pH, and the vertical dimension represents Mr.

20

Figure 2.2 Silver-stained 2-D gels of soluble proteins from primary polyps of Heteroxenia fuscescens, differing in their ages and symbiotic states: (A) 3-week-old aposymbiotic polyps. (B) 3-week-old symbiotic polyps, 1 week after infection with zooxanthellae. (C) 4-week-old aposymbiotic polyps. (D) 4-week-old symbiotic polyps. 2 weeks after infection with zooxanthellae. (E) 6-week-old aposymbiotic polyps. (F) 6-week-old symbiotic polyps. 4 weeks after infection with zooxanthellae. Triangles surround spots that intensify with age; inverted triangles - those that decrease in expression with age; rectangles - those that intensify only in the polyp stage. The horizontal dimension represents pH, and the vertical dimension represents Mr.

21

Figure 2.3 Summary of changes in spot intensity through host ontogeny. Enlargement of the spots marked on Figure 3.1.

2.4 Discussion

The present comparative proteomic study revealed changes in the host soft coral

proteome through development but surprisingly virtually no changes in the host proteome

as a function of symbiotic state. This suggests that during the first days and weeks of

symbiosis between Heteroxenia fuscescens and Symbiodinium sp., translational and post translational changes specific to the symbiotic state may not be occurring in the host.

These findings are remarkably similar to a recent comparative proteomic study of symbiotic and aposymbiotic larvae of the stony coral Fungia scutaria (deBoer, 2004; deBoer & Weis, in preparation). Two dimensional PAGE patterns of host proteins revealed only one consistent difference out of approximately 450 proteins between symbiotic and aposymbiotic larvae of the same age.

22 Our findings are, however, in stark contrast to those obtained from other comparative

proteomic studies of symbioses. In work comparing symbiotic and aposymbiotic

specimens of the temperate sea anemone Anthopleura elegantissima, numerous proteins

were shown to be specific to or enhanced in the symbiotic state (Weis & Levine, 1996).

Two abundant proteins were further characterized and identified as carbonic anhydrase

an enzyme known to function in inorganic carbon transport in cnidarian/algal symbioses

(Weis 1991; Al-Moghrabi et al., 1996; Weis & Reynolds 1999; Furla et al., 2000) and sym32, a fasciclin I homolog that may function in host/symbiont communication

(Reynolds et al, 2000; Schwarz & Weis 2003). A 2D protein profile study of the squid

Euprymna scolopes compared the changes in the soluble proteome of the symbiotic bacterial light organ during the first 96 h of symbiosis in symbiont-colonized and uncolonized organs (Doino Lemus & McFall-Ngai, 2000). Numerous symbiosis-related differences were found at 48 and 96 h after the onset of symbiosis and these changes were more abundant than age-related changes. Multiple studies using 2D PAGE and other techniques on the leguminous plant/nitrogen-fixing bacteria symbiosis have resulted in the characterization of a suite of plant genes specifically expressed during onset of symbiosis (Gloudemans & Bisseling 1989; Govers et al., 1985; Natera et al.,

2000; Saalbach et al. 2002). In addition, the onset of pathogenic associations has also been shown to causes changes in patterns of host proteins (e.g. Abshire & Neidhardt,

1993; Kwaik et al., 1994).

There are several other explanations for the uniformity of proteome patterns between symbiotic and aposymbiotic hosts. It is possible that changes are occurring but they are not being detected. This could be due to a variety of factors including slow protein

23 turnover with age of the host proteome, transient expression of symbiosis-specific or

enhanced proteins, and expression of these proteins in very low quantities below

detection levels. If symbiosis-specific protein expression is indeed limited to those cells

housing the symbionts, then these host cells number only in the hundreds to thousands in

the early weeks of the symbiosis (Yacobovitch 2001). In a background of many

thousands of cells comprising a juvenile polyp, any symbiosis-specific protein signal

might be lost. An example that further demonstrates this possibility is in E. scolopes,

where a differential result was obtained only in its symbiosis-specific organs that had

been dissected away from the rest of the animal (Doino Lemus & McFall-Ngai, 2000).

Since in Cnidaria algal symbionts are not housed in such a specific location, detecting

these symbiosis-specific changes is made difficult by a high non-symbiosis cell

background.

In conclusion, the onset of symbiosis between the soft coral H. fuscescens and

Symbiodinium sp. is not accompanied by gross changes in the host proteome in the first weeks of the association. Further studies employing more sensitive techniques, such as cDNA microarrays, are needed to identify proteins involved in the initial interactions between the partners.

24 SECTION III

Diversity of dinoflagellate symbionts in Red Sea soft corals:

mode of symbiont acquisition matters

3.1 Introduction

Among the most significant marine mutualisms are those found between members of the

phylum Cnidaria, such as corals and anemones, and their photosynthetic dinoflagellate

symbionts Symbiodinium spp, (also called zooxanthellae), which together form the

trophic and structural foundations of coral reef ecosystems. There is a rich literature on

coral systematics and phylogeny, based on skeletal morphology (Veron 1995) and more recently also on molecular markers (Chen et al., 2002). In contrast, classical taxonomic

studies of the zooxanthellae are hindered by what seems to be a uniform algal orphology.

The advent of molecular tools has resulted in the birth of the field of zooxanthellae

molecular phylogenetics and systematics. Zooxanthellae are now known to be

taxonomically highly diverse and have been divided into 5 distinct clades (A, B, C, D

and E), based on ribosomal RNA gene sequences and RFLP patterns (Rowan & Powers

1991a,b; Rowan, 1998; LaJeunesse, 2001; Toller et al., 2001). These clades represent

subgeneric taxonomic levels, and each contains 1 or more subtypes, as revealed by

analysis of the internal transcribed spacer regions (LaJeunesse, 2001; Savage et al.,

2002). Symbiotic systems, being based on the relationship of 2 different entities have

always been subject to the study of specificity (see Douglas, 1994). Dealing with

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) dual mechanism of symbiont acquisition— source of symbionts varies with host taxon; (3) the

25 fact that there are hundreds of host species and unknown numbers of symbiont species.

There are different aspects (biotic and abiotic) to the study of specificity and many of them have already been dealt with (see Baker, 2003). The use of finer resolution molecular tools and the screening of a wide array of cnidarian hosts over a wide biogeographic range (LaJeunesse, 2002; LaJeunesse et al., 2003) is doubtless of great importance to our understanding of specificity in this symbiosis. Another important factor to consider in studies of host-symbiont specificity is the mode of symbiont acquisition by the host (Rowan, 1998). The onset of symbiosis can occur at a variety of host life history stages, depending on the host species. Symbionts can be transmitted horizontally, where the host’s sexual progeny acquire symbionts from the surrounding environment, or vertically, being passed directly from host parent to offspring (Trench, 1987; Douglas,

1994). Horizontal transmission offers the host the opportunity to recombine with different algal types that are differentially adapted to the existing environmental conditions. There is a risk, however, that a host may fail to establish a partnership, leaving it with severely reduced fitness. In contrast, vertical transmission guarantees that a host is provided with a complement of symbionts. It is still unclear how the mode of symbiont acquisition influences zooxanthellae diversity within a host (Muller-Parker & D’Elia 1997;

Rowan, 1998). Soft corals (Octocorallia, ) are a significant component of the coral reefs of the northern Red Sea (Benayahu, 1985). The life histories and reproductive biology of many Red Sea soft corals are known, as well as the different modes of algal acquisition by the different species (Benayahu, 1997). To date, most studies examining zooxanthellae diversity in the Octocorallia have focused on seawhips (family:

Plexauridae) (Coffroth et al., 2001, Santos et al., 2001, LaJeunesse, 2002), while no comprehensive data exist on soft coral symbionts. Furthermore, data concerning

26 zooxanthellae diversity in Red Sea cnidarians are anecdotal (Goulet & Coffroth 1997;

Carlos et al., 1999; LaJeunesse, 2001). We compared symbionts found in hosts belonging

to the 3 most common soft coral families, which exhibit different modes of symbiont

acquisition, using restriction fragment length polymorphism (RFLP) of the 18S rRNA

gene (Rowan & Powers 1991b). Our results show a novel pattern of symbionts’ clade

segregation corresponding to the host’s mode of symbiont acquisition.

3.2 Materials and Methods

3.2.1 Collection and identification: Of the 3 families , Nephteidae and

Xeniidae, 19 soft coral species were investigated in this study (see Table 3.1). Samples were collected by SCUBA diving from sites along Eilat’s reefs (northern Red Sea) during

August 2001. From each species, 3 colonies were frozen and stored at –20°C until further analysis. Before freezing, a portion of each sample was preserved in 70% ethanol for species identification using the reference collection of the Zoological Museum of Tel Aviv University. When present, mature oocytes were examined under a compound microscope to detect the presence of zooxanthellae, in cases where no previous data were available.

3.2.2 Extraction of DNA: Samples were thawed, transferred to microfuge tubes, and ground with a small plastic pestle in filtered seawater. Homogenates were then centrifuged for 2 min at 2000 rpm (350 × g) to pellet the algae. Animal supernatant was removed and discarded and the algal pellet was resuspended in DNA extraction Buffer

(4M NaCl, 50mM EDTA, pH = 8.0). After further centrifugation, the pellet was resuspended in CTAB buffer and incubated for 2 h at 50°C together with Proteinase K.

The DNA extraction procedure was continued using a DNAeasy Tissue Kit (QIAGEN).

27 3.2.3 PCR amplification and restriction fragment length polymorphism (RFLP) analysis: The 18S rRNA gene was amplified from symbiotic dinoflagellates using the primers rRNA-F2 5’-TATTTGATGGTYRCTGCTAC-3’ and rRNA-R2 5’-

CRAATWATTCACCGGATCAC-3’, similar to those used by Rowan & Powers (1991a).

Amplifications were performed using a DNA thermal cycler (UNO II, BIOMETRA) under the following conditions: 94°C for 45 s, 54°C for 45 s and 72°C for 2 min (31 cycles). RFLP analysis was performed using restriction digests with Taq I (MBI

Fermentas) and Dpn II (BioLabs) restriction enzymes for 2.5 h at 65 and 37°C, respectively. Digestion products were separated by electrophoresis in 2% 0.5 X Tris-

Borate (TBE) agarose gels to generate RFLP patterns, which were compared to the literature to assign each sample to one of the established Symbiodinium 18S-rDNA RFLP clades (Rowan & Powers 1991a,b; Banaszak et al., 2000).

3.2.4 DNA sequencing: PCR products were cloned into pDrive cloning vector with

QIAGEN EZ competent cells as a host, using QIAGEN PCR Cloning Plus kit according to the manufacturer’s instructions. Plasmid DNA from individual colonies was purified using QIAprep Spin Miniprep kit (QIAGEN). The plasmid DNA was sequenced using the ABI PRISM BigDye Terminators v 3.0 Cycle Sequencing Kit (Applied Biosystems).

Due to the large size of the insert (1600 bp), each sample was sequenced from both sides

using T7 and SP6 primers. Partial sequences of symbiont genotypes derived in this study

were submitted to GenBank. Accession numbers for sequences of symbionts isolated

from the corals Litophyton arboreum, Rhytisma fulvum fulvum, Nephthea sp., Cladiella

tuberculoides, Sinularia querciformis, Sarcophyton glaucum, Xenia farauensis,

Paralemnalia eburnean, Stereonephthya cundabiluensis and Anthelia glauca are

AY525018–27 respectively. The symbiont isolated from Heteroxenia fuscescens is

28 available under the accession number AY488089. In order to sequence the second

genotype of symbionts, observed in 5 of the Alcyoniid corals, the PCR product of

the coral Sarcophyton glaucum was cloned as described above. The transformed bacteria

colonies were picked and used as template in a PCR reaction, using the vector primers.

PCR products were run on an agarose gel to insure an insert in the right size. ubsequently, the products were digested with Taq I restriction enzyme. Three PCR products, which showed the distinct band pattern, were sequenced as described above.

3.2.5 Phylogenetic analysis: Fragments of ssrRNA sequences were obtained for the 3’ and 5’ ends. These sequences were joined to form a partial composite sequence for each species. Sequences were aligned using the multiple sequence alignment program

CLUSTAL-XW (Thompson et al., 1997). The following reference sequences taken from

GenBank for the 18S rRNA gene were also included in the alignment: Symbiodinium sp.

GenBank accession number AF238256 (Clade A); Symbiodinium sp. GenBank accession number AB016594 (Clade C); and Gymnodinium simplex GenBank accession number

GSU41086 (Outgroup). The alignment was checked and adjusted by eye using the sequence manipulation program MacVector. Only the aligned sequence portions which were represented by all sequences included were used in the phylogenetic analysis. This was performed by excluding non-overlapping characters using the mask tool of

MacVector. Gaps in the alignments were treated as a fifth character. A total of 1114 characters were included in the mask. The final alignment with the applied mask was converted to a Nexus format file and used with the phylogenetic analysis program

PAUP* 4.0. The phylogenetic reconstruction was performed with the Neighbor Joining

(NJ) applying the Kimura 2-parameter model, as well as Maximum Parsimony method.

The sequence from Gymnodinium simplex (GenBank accession number GSU41086) was

29 used as the outgroup. Bootstrapping was performed using 1000 replications and a

Bootstrap consensus tree was constructed using the 50% majority rule.

3.3 Results

Among the 19 soft coral species studied (Table 3.1), 16 contained zooxanthellae

belonging to Clade C, whereas 3 species contained zooxanthellae from Clade A (Figs. 3.1

& 3.2). All species harboring Clade C algae acquire their symbionts horizontally from the

environment, while the species harboring Clade A algae acquire theirs directly from the

parent at the oocyte stage. The RFLP patterns of 5 of the alcyoniid corals revealed an

additional faint band 720 bp in size (Fig 3.1, Lanes 9 to 13). This band is indicative of the

presence of another symbiont genotype. Cloning and sequencing of this genotype

(isolated from Sarcophyton glaucum) revealed that it belongs to Clade C. The mode of

sexual reproduction in soft corals (families Alcyoniidae, Nephteidae and Xeneiidae) and

features such as the onset of symbiosis during development and presence or absence of

zooxanthellae in oocytes exhibit a high degree of consistency within genera (Benayahu,

1997). Thus, in the few cases in our study where no data are provided for a given species

in Table 3.1, it is likely that the mode of reproduction, presence/absence of zooxanthellae

in oocytes and stage of algal acquisition follow the respective patterns of the congeners.

All the Alcyoniidae corals studied, including spawners and a surface brooder (Table 3.1), acquired algae from the environment during their primary polyp stage (Table 3.1)

(Benayahu, 1997) and possessed zooxanthellae belonging to Clade C (Fig. 3.1, Lanes 1 to

8) or a combination of 2 genotypes of Clade C (Fig. 3.1, Lanes 9 to 13). Similarly, all

Xeniidae examined possessed Clade C zooxanthellae. The latter display a diversity

30 of brooding mechanisms and differ in the developmental stage during which symbionts are acquired. Planulae of Heteroxenia fuscescens lack zooxanthellae when released and acquire symbionts soon after metamorphosis (Benayahu et al., 1989a,b). In Anthelia glauca, zooxanthellae appear in the embryos, which are brooded within the pharyngeal cavity of the polyps (Benayahu & Schleyer 1998, Kruger et al. 1998). Planulae of Xenia species develop inside invaginated brooding chambers lined with ectoderm, which are open to the environment and contain zooxanthellae that are phagocytosed by the brood

(Achituv et al., 1992). Hence, planulae of A. glauca and Xenia species acquire their symbionts horizontally prior to release. Thus, all the studied alcyoniid and xeniid soft corals, despite variations in the mode of reproduction and stage of acquisition (see also

Table 3.1), contain symbiotic algae belonging to Clade C. It is in the Nephtheidae that the different modes of symbiont acquisition were found to correlate with the symbiont clade found in adults. Although all 5 nephtheid species studied are brooders, the 2 in the genus

Paralemnalia (P. thyrsoides and P. eburnea), release azooxanthellate planulae (Table

3.1) that must acquire symbionts horizontally. Like the above-mentioned xeniid and alcyoniid species with horizontal transmission, these too contained Clade C zooxanthellae. In contrast, Litophyton arboreum, Nephthea sp. And Stereonephthya cundabiluensis have vertical symbiont acquisition, where zooxanthellae are directly transmitted to their sexual progeny at the oocyte stage (Table 3.1). These 3 species uniquely harbored Clade A zooxanthellae.

31 Table 3.1 List of soft corals examined, their modes of reproduction and developmental stage during which symbionts are acquired. * partial sequence of 18S rRNA gene of zooxanthellae is available, n.d = no data

Species Mode of Zooxanthellae in Stage of acquisition Reference reproduction oocytes Alcyoniidae

Cladiella pachyclados Spawning No Primary polyp This study

Cladiella tuberculoides* n.d. n.d. n.d.

Rhytisma fulvum fulvum* Surface No Primary polyp Benayahu & Loya 1983 brooding Sarcophyton glaucum* Spawning No Primary polyp Benayahu & Loya 1986

Sarcophyton Spawning No Primary polyp Shinkarenko 1981, work in trocheliophorum progress

Sinularia gardineri n.d. No n.d. This study

Sinularia leptoclados Spawning No Primary polyp Benayahu et al. 1990

Sinularia polydactyla Spawning No Primary polyp Alino & Coll 1989

Sinularia querciformis* n.d. No n.d. This study

Xeniidae

Anthelia glauca* Pharyngeal No Embryo Kruger & Schleyer 1998 brooding Heteroxenia fuscescens* Brooding No Primary polyp Benayahu 1991

Xenia farauensis* Brooding No Embryo Benayahu & Loya 1984, work in progress Xenia macrospiculata Brooding No Embryo Benayahu et al. 1992

Xenia umbellata Brooding No Embryo Benayahu 1991

Nephtheidae

Litophyton arboreum* Brooding Yes Oocyte Benayahu et al. 1992

Nephthea sp.* Brooding Yes Oocyte Benayahu 1997, Lutzky 1997

Paralemnalia eburnea* n.d. n.d. n.d.

Paralemnalia thyrsoides Brooding No n.d. Benayahu 1997

Stereonephthya Brooding Yes Oocyte Benayahu 1997; This study cundabiluensis*

32

Figure 3.1 Taq I RFLP analysis of ss-RNA encoding DNA of zooxanthellae isolated from different Red Sea soft corals. The list of lanes indicates the names of the corals from which algal DNA was extracted. Lane 1 Anthelia glauca, lane 2 Heteroxenia fuscescens, lane 3 Xenia farauensis, lane 4 Xenia macrospiculata, lane 5 Xenia umbellata, lane 6 Cladiella tuberculoides, lane 7 Cladiella pachyclados, lane 8 Sinularia querciformis, lane 9 Sinularia polydactyla, lane 10 Sinularia leptoclados, lane 11 Sinularia gardineri, lane 12 Sarcophyton glaucum, lane 13 Sarcophyton trocheliophorum, lane 14 Rhytisma fulvum fulvum, lane 15 Paralemnalia eburnea, lane 16 Paralemnalia thyrsoides, lane 17 Litophyton arboreum, lane 18 Nephthea sp., lane 19 Stereonphthya cundabiluensis, lane 20 1kb Plus DNA ladder (GibcoBRL).

33

S. sp (Anthelia glauca)

S. sp. (Heteroxenia fuscescens) S. sp (Xenia 69

S. sp (Paralemnalia eburnea) (70) S. sp. (Sarcophyton glaucum)

S. sp. (Sinularia querciformis) Clade C

10 64 S. sp. (Cladiella tuberculoides) (100) (57) S. sp. (Rhytisma fulvum Symbiodinium sp. (GenBank accession #:

98 S. sp. (Litophyton arboreum)

79 (98) S. sp. (Nephthea sp.) 100 (62) S. sp. (Stereonephthya cundabiluensis) (100) Symbiodinium microadriaticum. Clade A (GenBank accession #: M88521)

Gymnodinium simplex

Figure 3.2 Phylogenetic reconstruction of Symbiodinium spp. from different soft coral host species using partial sequences of 18s rRNA gene. Trees constructed based on both the NJ and MP methods had the same topology. The names in parentheses indicate host species from which algal (symbiont) sequences were obtained. Gymnodinium simplex was used as the outgroup. The Bootstrap consistency values >50 based on 1000 replicates are shown above the branches for the NJ analysis and under the branches in parentheses for the MP analysis.

2.4 Discussion

Symbiotic associations between Cnidarian hosts and their symbiotic algae exhibit two possible modes of symbiont acquisition: vertical and horizontal. We examined multiple host species in one location and herein provide, for the first time, perspective on the mode of symbiont acquisition and its relation to clade specificity in a variety of soft coral

34 hosts. Previous studies addressing this subject in stony corals have examined a restricted number of species over a broad biogeographic scale (Hidaka & Hirose 2000; Loh et al.,

2001; Rodriguez-Lanetty et al., 2001). Recently, Van Oppen (2004) compared the iversity of symbionts in 25 Montipora species (vertical transmission) and Acropora species (horizontal transmission) in Indonesia and the central Great Barrier Reef,

Australia. They found that the mode of symbiont acquisition does not affect symbiont diversity (i.e. number of symbiont types within a host) within acroporid corals. The Gulf of Eilat, situated at the northernmost limit of coral reefs distribution, is characterized by extreme environmental conditions such as catastrophic low tides, elevated temperatures and high irradiance (Loya, 1986; Achituv & Dubinsky, 1990). However, no bleaching events have been reported from this area (Pilcher & Alsuhaibany, 2000). Our data indicate that hosts harboring either Clade A or Clade C symbionts co-occur in the same habitats. For example, Litophyton arboreum (Clade A) and Rhytisma fulvum fulvum (Clade C) form monospecific carpets on Eilat’s reef flats (Benayahu & Loya, 1977). Furthermore, molecular analysis of zooxanthellae from Heteroxenia, Sinularia, Rhytisma,

Stereonephtya and Litophyton, sampled over a depth gradient (1 to 20 m), reveals persistence in clade specificity with depth within a host (O. Barneah unpubl. data).

Therefore, the distribution of symbiont clades in Eilat’s soft corals negates the correlation between symbiont clade and depth demonstrated in Caribbean reefs (Rowan & Knowlton

1995; Rowan et al., 1997; LaJeunesse, 2001; Toller et al. 2001). Symbionts belonging to

Clade A were only found in hosts that vertically transmit their symbionts. There is evidence from studies performed in the Caribbean that Clade A zooxanthellae are shallow-water specialists (Toller et al., 2001, LaJeunesse, 2002), and are relatively

35 stress tolerant or ‘weedy’ (Rowan, 1998). It has also been shown that Clade A

zooxanthellae are widely tolerant to temperature change (Kinzie et al., 2001) and are the

only clade capable of synthesizing mycosporine- like amino acids (MAAs) (Banaszak et

al., 2000). Together, these studies suggest that Clade A algae may be well adapted to

cope with Eilat’s environmental conditions. We suggest that Clade A symbionts, evolved

to be optimal vertically transmitted symbionts, that did not fail their host and thus

persisted

as faithful symbionts for generations. It should be pointed out that data are lacking concerning the survival of Clade A symbionts in the free living state. For example, they might be very successful within the coral tissues but outcompeted by different algal clades while in the water column. The limitation of Clade A symbionts to hosts with vertical transmission suggests a coevolution of the hosts and symbionts. Data on the phylogeny of Red Sea soft corals that could be overlaid onto the phylogeny of the symbionts will yield further information on how the observed specificity pattern arose. Were Clade A algae ‘captured’ by a single nephtheid ancestor that gave rise to the

3 vertically transmitting genera, or did this event occur a multiple of times within the family? No congruence was found between Montipora host and symbiont phylogenies, a fact that might be attributed to occasional lateral transfer of zooxanthellae between

species, which might have occurred as a result of interspecific hybridization

arising during mass spawning events (Van Oppen et al., 2004). We anticipate a different

result in the 3 nephtheid hosts harboring Clade A zooxanthellae. These corals are

brooders, from a region characterized by temporal reproductive isolation rather than by

mass spawning events (Shlesinger & Loya, 1985), representing the most conservative

36 scenario among cnidarians, in terms of vertical transmission. Clade C symbionts, characterized by large sub-clade variability (Fig. 3.2), are found in corals with horizontal transmission and, most probably, each of this clade’s genotypes exhibits a more specialized set of physiological capabilities. Recently, LaJeunesse et al. (2003) found that the majority of endosymbiotic dinoflagellates in cnidarians in the Great Barrier Reef

(Australia) belong to Clade C, which is composed of closely related, yet ecologically and physiologically distinct, types. The observed combination of 2 different genotypes of

Clade C in 5 species of alcyoniid soft corals further highlights the flexibility of an open system of symbiont acquisition. We hypothesize that free-living stages of Clade C symbionts are prevalent in the reef area and are available for acquisition by juvenile stages of soft coral hosts. Future studies examining distribution and availability of symbionts in the open water and physiological plasticity of different symbionts strains are needed to test these hypotheses. Based on our results from soft corals and on the recent literature (LaJeunesse et al., 2003; Van Oppen, 2004a) in stony corals (Montipora and Porites respectively), we hypothesize that corals which vertically transmit their symbionts will tend to have genetically distinct symbionts, which differ from those found in corals with horizontal transmission. In our study, the distinctive feature is symbiont clade, while in Montipora and Porites, the symbionts were found to belong to a distinct sub-clade. Whether the symbionts belonging to Clade A (from the 3 studied nephtheid corals) represent one type or 3 distinct, yet similar, types is yet to be determined. Vertical transmission is relatively rare among cnidarians (Trench, 1987). Coral hosts with vertical transmission of symbionts are assumed to represent a scenario in which a certain ancestral symbiont was ‘trapped’ and evolved within host tissues. It is likely that these

37 holobionts were exposed to changing environmental conditions during their evolution and their survival therefore indicates their resilience. Is it possible that corals which vertically transmit their symbionts are more resistant to environmental change? Are coral hosts with vertical transmission less susceptible to bleaching? There are some data in support of these hypotheses. Among a variety of Great BarrierReef stony corals, 2 of the most bleaching resistant species were Montipora digitata and Porites cylindrica, which both transmit symbionts vertically and associate with the unique Symbiodinium strain C15

(LaJeunesse et al., 2003). A combined comparative approach of sampling symbionts from hosts with vertical versus horizontal transmission, and analysis of their physiological capabilities, can answer these questions. New data are continually emerging on zooxanthellae systematics and the relation thereof to host-symbiont specificity. Our study, by examining the correlation between symbiont taxon and the mode of symbiont acquisition by soft coral hosts, describes another layer of complexity in this symbiotic relationship. The pattern of clade distribution which we describe is exceptional, and raises many questions to be addressed on a global scale. Looking at other symbiotic systems, such as the aphid-bacteria, in which the phylogeny of symbionts parallels their hosts (Douglas, 1994), it is very clear that we are uncovering a multidimensional concept of specificity, rather then a 2-dimensional one.

38 SECTION IV

Interactions involving acoelomorph worms, corals and

dinoflagellate algal symbionts in Eilat (Red Sea)

4.1 Introduction

Coral reefs are often described as the rain forest of the sea (Connell, 1978). As such, they

include numerous organisms, which interact with each other in a complex array of

symbiotic associations (Paulay, 1997). A single coral colony may accommodate a variety

of symbiotic organisms including invertebrates and vertebrates, bacteria, and algae, all

living in close proximity and using different resources from their host or from each other

(Paulay, 1997).

The most crucial of these symbioses involves invertebrates and photosynthetic

dinoflagellates (Trench, 1992). In this system the potential hosts may belong to members

of various phyla including Porifera, Cnidaria, Platyhelminthes and Mollusca (Douglas,

1994), as well as Acoelomorpha, a recently designated phylum (Baguna & Riutort, 2004).

Cnidarian-algal symbiosis is currently being intensively studied especially in light of the

frequent coral bleaching episodes occurring worldwide (Hoegh-Guldberg, 1999).

Contrary to the well-studied cnidarians, symbiotic platyhelminth worms and particularly

acoelomorphs are the most understudied group of associations considering the numerical abundance, diversity of taxa, range of habitats, and their geographic distribution (McCoy

& Balzer, 2002). Recent molecular and morphological evidence suggests that members of

39 the phylum Acoelomorpha are the most basal triploblastic Bilateria (Baguna & Riutort,

2004; Ruiz-Trillo et al., 2004). This phylum includes the classes Nemertodermatida and

Acoela. The latter, the subject of our study, is a morphologically diverse group of small

(~0.5-10 mm long) and soft-bodied worms (Hooge et al., 2002), lacking a gut cavity and

protonephridia (Tyler, 2003). To date, there are over 340 known acoel species assigned to

21 families (Tyler et al., 2005).

Three of the families contain species with symbiotic unicellular algae (Convolutidae,

Sagittiferidae, and Haploposthiidae) (McCoy & Balzer, 2002). The algae that may form a symbiosis with acoels belong to separate lineages, including members of the

Bacillariophyta (diatoms), Dinophyta (dinoflagellates) and Chlorophyta (green algae)

(see McCoy & Balzer, 2002). Among acoels, three of the well-documented symbiotic associations are those of Symsagittifera roscoffensis with the prasinophyte Tetraselmis,

Convoluta convoluta with the diatom Licomophora, and Amphiscolops with the

dinoflagellate Amphidinium (Douglas, 1992). Symbiotic acoel worms that are epizoic on

corals include Haplodiscus sp. from reefs near Micronesia (Trench & Winsor, 1987),

Waminoa litus, Waminoa sp. 1, 2 and Convolutriloba hastifera from North Queensland,

Australia (Winsor, 1990). Haplodiscus sp. contains two distinct algal symbionts within

the same host cell. This worm was described as pelagic, but spends part of its lifecycle on

the stony coral Porites (Trench & Winsor, 1987). Waminoa litus was found living on the

soft coral Sarcophyton from Magnetic Island, Australia and contains two species of algal

symbionts identified as Symbiodinium sp. (8 μm in diameter) and Amphidinium sp. (16-

24 μm in diameter) (Winsor, 1990). Waminoa sp. 1 and 2, collected from un-specified

corals in the marine aquarium of the Australian Institute of Marine Science (AIMS) and

from the stony coral Acropora longicyathus from Pandora reef, Australia, respectively,

40 were associated with two different-sized algal symbionts (Winsor, 1990). Convolutriloba hastifera was collected from a soft coral in the aquarium at AIMS; its unidentified symbionts measured 7-12 μm (Winsor, 1990). Interestingly, coral reef aquarium hobbyists often encounter infestations by these worms. They report that the worms have been observed on damaged corals, feeding on their tissues and incorporating the algal symbionts (Delbeek & Sprung, 1994).

Most studies on symbiotic acoels address the morphology of the worms (Winsor, 1990;

Trench & Winsor, 1987). The nature of the symbiotic association i.e., the possible benefits or disadvantages of each of the three associated partners - namely worms, corals and algal symbionts, is relatively unexplored.

The occurrence of Symbiodinium in both corals and worms (this work) and the close physical contact of the worms with their coral hosts gave rise to the following working hypothesis: Waminoa sp. worms acquire their dinoflagellate algal symbionts from their coral hosts. Testing this hypothesis with genetic methods will shed light on the nature of the symbiotic interaction between corals, worms and dinoflagellate algae i.e. it will clarify whether the worms feed on the coral tissue and incorporate its algal symbionts.

The present section examines the diversity of coral hosts harboring acoel worms in

Eilat, worms’ morphology, the structure of their symbionts, the possible effects of the worms on their coral hosts, and compares the genetic identity of Symbiodinium spp. populations living in corals and in their epizoic worms.

41

4.2 Materials and Methods

4.2.1 Field observations and collection of animals: Field-work was performed by

SCUBA diving on three reef patches in Eilat, including the reef across from the Inter

University Institute (IUI), near the underwater observatory and at the oil jetties at a depth

range of 2-50 meters. Our observations indicated that seven species of stony corals most

commonly harbor the worms (see Results). In order to examine the abundance of the

worms on a given coral host, surveys were conducted during August 2004 at the IUI along a 70 meter belt transect at 3 depth zones: 2-3, 7-8 and 18-20 m. Each colony of

these seven species was counted carefully and checked for the presence of worms.

Acoelomorph worms were collected during May, July, October and December 2003 and

February and March 2004. Worms were collected with a fragment of their host colony,

placed into a plastic container and kept in running sea water at the IUI until further

observations and analysis. Four coral species were chosen for in-depth study. These

included the stony corals Acropora hemprichi Ehrenberg, 1834 Plesiastrea laxa,

Klunzinger, 1879, Stylophora pistillata, Esper, 1797 and the soft coral Stereonephthya

cundabiluensis, Verseveldt, 1965. In addition, worms were removed from A. hemprichi,

P. laxa and S. pistillata (n=10 from each coral host), preserved in 2.5% glutaraldehyde in

sea water and measured under a dissecting microscope.

4.2.2 Scanning electron microscopy of worms: In order to study the morphological

features of the worms we removed 10 worms from each of the four above-mentioned

coral hosts. Specimens were preserved in 2.5% glutaraldehyde in sea water, dehydrated

through a graded series of ethanol concentrations, critically point-dried with liquid CO2,

coated with gold and examined under a JEOL JSM 840 scanning electron microscope.

42 4.2.3 Scanning electron microscopy of coral surface: In order to compare the surface

of infested coral hosts vs. non-infested one, we cut branches (3 cm in length) from

colonies of S. cundabiluensis, with and without worms. Worms were removed from the

respective branches using a mild water stream produced by a plastic pipette, and the two

types of fragments were preserved and prepared for SEM as described above.

4.2.4 Transmission electron microscopy of worms: To study the structure of the worms

and features of their symbionts, as well as the position of the latter inside their host, worms preserved in 2.5% glutaraldehyde in sea water were rinsed in buffer phosphate, stained with 1% OsO4 , dehydrated through a graded ethanol series and embedded in

Epon. Sections were cut with a diamond knife, stained with lead citrate, and viewed with

JEOL 1200 EX transmission electron microscope.

4.2.5 Genetic analysis of Symbiodinium spp. symbionts from corals and resident

worms: Coral fragments containing worms from 3-6 different colonies of Turbinaria sp.,

Plesiastrea laxa, Acropora hemprichi and Stylophora pistillata and the soft coral

Stereonephthya cundabiluensis were brought to the laboratory alive. Worms (30-50 individuals) were separated from the coral fragment using a plastic pipette and transferred to microfuge tubes and subsequently frozen. In order to obtain the algal symbionts from the coral tissue, the latter was stripped from the skeleton using a scalpel, homogenized with a plastic pestle, and then centrifuged for 10 min at 2000 rpm to pellet the algae.

4.2.6 Extractions of zooxanthellae DNA: DNA extractions were performed using

Invisorb Spin Plant Mini Kit (Invitek) according to the manufacturer instructions. Algal pellets derived from coral tissues were ground in a microfuge tube with a small plastic pestle in the presence of lysis buffer. Entire worm specimens were ground and total genomic DNA was isolated from both the worm and their symbionts.

43 4.2.7 Denaturing-gradient gel electrophoresis (DGGE): PCR-DGGE was used to analyse the ITS2 of nuclear ribosomal RNA genes (LaJeunesse 2001, 2002). PCR-DGGE analyses were conducted using the forward primer “ITSinfor2” (5’-GAATTGCAGA

ACTCCGTG-3’) (LaJeunesse & Trench, 2000), which anneals to a “Symbiodinium- conserved” region in the middle of the 5.8S ribosomal gene and the highly conserved reverse primer that anneals to the LSU “ITS2CLAMP” (5’-CGCCCGCCGC

GCCCCGCGCC CGTCCCGCCG CCCCCGCCC GGGATCCATA TGCTTAAGTT

CAGCGGGT-3’), an ITS-reverse universal primer modified with a 39-bp GC clamp

(underlined) (LaJeunesse & Trench, 2000). A “touchdown” amplification protocol with annealing conditions 10°C above the final annealing temperature of 52°C was used to ensure PCR specificity. The annealing temperature was decreased by 0.5°C after each of the 20 cycles. Once the annealing temperature reached 52°C, it was maintained at that setting for another 20 cycles. Samples were loaded onto an 8% polyacrylamide denaturing gradient gel (45%-80% urea-formamide gradient: 100% consists of 7 mol L-1 urea and 40% deionized formamide) and separated by electrophoresis for 9.5 h at 150 V at a constant temperature of 60°C (LaJeunesse 2002). The gel was stained with Sybr

Green (Molecular probes) for 25 min according to the manufacturer’s specifications and photographed using 667 Polaroid film.

4.3 Results

Field surveys revealed that 14 anthozoan species were infested by acoel worms in the depth range surveyed 2 to 50 meters. The 13 stony coral species that were infested belong to 6 different families (Acroporidae, Pocilloporidae, Faviidae, Mussidae, Siderastreidae,

Dendrophylliidae). All host species were zooxanthellate and included both massive and

44 branching stony corals and one soft coral (Table 4.1). These corals had variable worm

densities, ranging from colonies that were densely populated (completely covered,

appearing brown in color) by worms to others that were sparsely populated (containing

few dozens worms) . Turbinaria sp. Oken, 1815 and Echinophyllia sp. Klunzinger, 1879

showed the highest infestation rate (Table 4.1), yet colonies of these corals occurred

rarely. The common Red Sea coral Stylophora pistillata was mildly infested (Table 4.1).

Corals occupied by worms showed no visible surface damage or lesions.

Worms from all hosts were identified as belonging to the genus Waminoa, although a

species designation could not be made, as most of the specimens were sexually immature

(see Tyler, 2003).

Figure 4.1A-C presents images of A. hemprichi, S. pistillata and P. laxa infested by worms. Interestingly, worms appear all over the coral surface. In S. pistillata, I observed

expanded polyps during the daytime, surrounded by worms (Fig. 4.1B). In P. laxa worms

were present on the external and internal sides of the polyp's calyx (Fig. 4.1C). Worms

derived from A. hemprichi were 1.18±0.345 mm in length, from S. pistillata 1.14±0.13

mm and from P. laxa 2.63±0.397 mm (n=10 worms from each coral species). All worms

were characterized by a golden brown color, due to presence of algal symbionts. Their

dorsal surface contained white refractile bodies, evenly scattered. Worms removed from

A. hemprichi possess genital pores (Fig. 2A). The male pore is located at the ventral

posterior end of the animal, situated in the center of a swollen area, with the female pore

adjacent to it, but more anterior. In worms removed from S. pistillata and P. laxa no

genital pores could be seen (Fig. 4.2B, C).

45 Scanning electron micrograph of a fractured worm isolated from A. hemprichi shows dorsal and ventral surfaces, covered with cilia, 5-8 μm in length (Fig. 4.3). Algal symbionts are scattered inside the worm.

Table 4.1 Occurrence of acoelomorph worms on coral hosts in Eilat (Red Sea). Infestation ratio is given as number of infested colonies vs. number of colonies counted.

Species of host coral Depth range Infestation ratio Acropora hemprichi 12-18 4/61 Echinophyllia sp. 2-26 1/5 Favia favus 6-10 6/74 Favites pentagona 8-13 3/40 Plesiastrea laxa 8-21 6/34 Stylophora pistillata 6-40 5/225 Turbinaria sp. 6-50 2/10 Acantastrea echinata 3-21 * Cyphastrea sp. 12 * Echinopora sp. 22 * Montipora sp. 22-27 * Platygira sp. 20 * Siderastrea sp. 8-15 * Stereonephthya cundabiluensis 10-22 *

*Infested colonies rarely encountered

46

Figure 4.1 Stony corals colonized by Waminoa sp. worms. (A) Acropora hemprichi (B) Stylophora pistillata with worms surrounding expanded polyps during daytime. (C) Plesiastrea laxa. Scale bar: 3 mm.

Figure 4.2 Scanning electron microscope images of worms isolated from Red Sea stony corals. (A) A ventral view of a specimen from Acropora hemprichi with a male gonopore (black arrow) and female gonopore (white arrow). (B) Specimen from Stylophora pistillata with no visible openings. (C) Specimen from Plesiastrea laxa. Scale bar: 100 μm.

47

Figure 4.3 Scanning electron micrograph of a fractured acoelomorph worm. Layer of cilia 5-8 μm in length covers the dorsal (white arrows) and ventral (white arrow heads) surfaces. Numerous small algal symbionts (white asterisk) and a few larger ones (black asterisk) are scattered in the parenchyme. Scale bar: 10 μm.

Fig. 4.4A-C present TEM micrographs of the algal symbionts within the worm host. All

the worms studied (removed from A. hemprichi, P. laxa, S. pistillata and S.

cundabiluensis) contained algal symbionts of two distinct sizes: small ones 5-8 µm and

larger ones 10-20 µm in diameter, with the former being much more abundant. Both

symbiont types are located within the worms’ parenchyme, each engulfed by cytoplasmic

processes (Fig. 4.4A). The epidermis is ciliated and the parenchymal cell nucleus is in

close proximity to the algal symbionts. The smaller symbiont, identified as Symbiodinium

sp. (Barneah et al., 2004) is characterized by a double-stalked pyrenoid that is not

penetrated by thylakoid membranes. The larger algal symbiont appears oval in shape,

with an irregular margin line containing surface clefts and ridges (Fig. 4.4B). The epicone

and the hypocone regions (terminology follows R.E Lee, 1999) of the cell are distinct as

is the theca, consisting of three layers of membranes (Fig. 4.4B). The nucleus, situated at

the posterior end of the hypocone, contains multiple condensed chromosomes (Fig.

48 4.4B). The pyrenoid, situated above the nucleus, shows no evidence of thylakoids

invasions. Chloroplasts are scattered around both the cell’s periphery and in its interior.

Fig. 4.4C presents a ‘larger’ symbiont with a flagellum within the sulcus, characterized

by ‘9+2’ arrangement.

Figure 4.4. Transmission electron micrographs of algal symbionts within Waminoa sp. (A) A section through a worm isolated from Acropora hemprichi. Epidermal cilia in various section orientations (asterisks). A nucleus of a parenchymal cell (nw) located beneath the muscle layer (m) in close vicinity to algal symbionts. On the left side an algal symbiont with a nucleus (nz) and chloroplast (cp). An arrow indicates the smaller symbiont with a double-stalked pyrenoid (py). Scale bar 2 μm. (B) The larger symbiont (Ampidinium sp.) from Waminoa sp. isolated from Stylophora pistillata with irregular margin line containing clefts and ridges. The epicone (e) and the hypocone (hy) regions of the cell are distinct. Dark arrows indicate the theca, consisting of three layers of membranes. The nucleus is situated at the posterior end of the hypocone (nz), containing 38 condensed chromosomes (c). Pyrenoid (py) is located just above the nucleus. Scattered chloroplasts (cp) appear around the periphery and the internal part of the cell. Scale bar 1 μm (C) Symbiont with a flagellum within the sulcus, with internal ‘9+2’ arrangement (insert). Scale bar 2 μm.

49 The surface of worm-infested Stereonephthya cundabiluensis had distinct surface

microvilli (Fig. 4.5A) while non-infested colonies possessed a layer of mucus (Fig. 4.5B).

Moreover, SEM images of worm-infested colonies indicated that the coral tissue did not

contain any lesions or injuries.

The Symbiodinium spp. from both the coral and the resident worm population were

analyzed using PCR-DGGE fingerprints. In general, the symbiont in the coral was not the

same as the symbiont found in the worm (Fig. 4.6). Acropora hemprichi and Plesiastrea

laxa shared the same Symbiodinium type, C41 (clade C type 41), while their resident

worms both had type C74. Turbinaria sp. possessed C1 while its worms had mixed

populations of two different types: C66 occurred consistently and usually in greater

relative proportion than type A11. The soft coral Stereonephthya cundabiluensis

possessed A9 while its worms possessed A11. Stylophora pistillata and its worms share

C72 symbionts but in a few replicates (from different colonies), the worm populations

also possessed A11 symbionts.

Fig. 4.7 presents the PCR-DGGE fingerprints of Symbiodinium spp. sampled from 6

colonies of the stony coral Turbinaria sp. and the fingerprints of symbionts from their resident worms. Four colonies (Tu2-5) harbored C1, 1 colony (Tu10) harbored C41 and another colony (Tu11) harbored both C1 and C41. Their worms all possessed symbionts belonging to type C66 (the migration of C66 during electrophoresis is similar to C1). In addition, the worms on one Trubinaria colony (Tu4) possessed A11 Symbiodinium in addition to C66.

50

Figure 4.5 Scanning electron micrographs of the surface area of the soft coral Stereonephthya cundabiluensis. (A) A fragment inhabited by Waminoa sp. appears clean of mucus, shown with protruding microvilli. (B) Fragment devoid of worms, covered with a mucus layer. Scale bar 1 μm.

Figure 4.6 Representative PCR-DGGE ITS2 fingerprints (profiles) of 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). Uppercase letters indicate lineage or clade, numbers represent ITS type. Examples of heteroduplexes are indicated; they are artifacts of the DGGE-PCR process present in fingerprints of genomes with more than 1 dominant ITS 2 sequence.

51

Figure 4.7 PCR-DGGE ITS2 fingerprints of Symbiodinium symbionts obtained from six 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 (indicated with the lowercase letter w).

4.4 Discussion

Numerous coral taxa surveyed near Eilat were found to possess, in some cases, dense

surface populations of acoel flatworms in the genus Waminoa, previously described from

Australia (Winsor, 1990). Sexually mature worms from the coral Plesiastraea laxa were

identified as a new species named Waminoa brickneri (Ogunlana et al., 2005). To date,

the only other described species in this genus is W. litus (Winsor, 1990). Worms isolated

from the corals Acropora hemprichi, Stylophora pistillata and Plesiastrea laxa exhibited

variability in morphological features, such as size and reproductive state, which might be

indicative for the presence of several species of Waminoa in Eilat. The classification of

52 acoelomorph species is based on characteristics of their reproductive organs (Tyler,

2003). Since most of the worms collected in the present study were not sexually mature,

species identification of worms from different coral hosts was not feasible, and therefore,

the specificity between coral host and resident worms cannot be resolved at this stage. To

date, molecular data concerning the genus Waminoa, which could be helpful as a

systematic tool is still scarce (Ogunlana et al., 2005). In the future, such data will surely

aid in the classification of these worms.

The lamellose coral species Turbinaria sp. and Echinophyllia sp. showed the highest infestation rate (Table 4.1). However, it should be noted that in these two species only one colony out of five was infested. On the other hand, in the branching coral S. pistillata, which is one of the most abundant stony corals in Eilat (Loya, 1972), only five out of 225 sampled colonies were infested. Overall, infestation rate appears to be low and patchy in nature. Hence, whereas one coral colony of a certain species may be found completely covered with worms, other colonies in its close vicinity (same species or different) may be worm-free. Our quantitative survey took place only at the reef across from the IUI. Undoubtedly, additional surveys along the northern Red Sea are needed in order to obtain a realistic figure of the extent of the worms' distribution and prevalence on various coral taxa.

Only worms from A. hemprichi contained genital openings. All the other specimens lacked such openings and even a mouth could not be detected. A low occurrence of sexually mature acoelomorph worms has been also encountered in other studies of

53 Waminoa sp. and Haplodiscus (Trench & Winsor, 1987; Winsor, 1990). Such findings suggest that sexual reproduction might be seasonal.

The worms contained two distinct algal symbionts. The smaller ones, which possessed a double-stalked pyrenoid (Fig. 4A) were assessed genetically using PCR-DGGE analysis of ITS-2 rDNA (LaJeunesse, 2001) and were assigned to the genus Symbiodinium. The larger symbionts were characterized by the presence of multiple chloroplasts, an irregular cell margin and the presence of a flagellum (Fig. 4C). The flagellum within the sulcus is most probably the longitudinal flagellum. The irregular margin line, characterized by the presence of clefts and ridges, is presumed to have resulted from section planes cutting through both flagellar canals i.e., the girdle and the sulcus. Several species of

Amphidinium are known to occur as symbionts in acoelomorph worms, including

Waminoa litus, Haplodiscus sp. (Winsor, 1990) and three different species of

Amphiscolops (Taylor, 1971; Trench & Winsor, 1987; Lopes & Silveira, 1994).

Interestingly, in symbiotic Amphidinium species, whether intercellular (Taylor, 1971) or intracellular (Trench & Winsor, 1987), the algae in hospite were found to retain their free-living morphology, including the flagellar apparatus, similar to our findings (Trench,

1993; this study Fig. 4.4C). Amphidinium klebsii symbionts from Amphiscolops langerhansi were reported to present a specific and uniform orientation within their host

(Taylor, 1971), a unique trait, that has not been reported in any other symbiotic acoelomorph including Waminoa sp. from Eilat. The symbionts from Amphiscolops langerhansi were characterized by the presence of a spherical pyrenoid, which formed the center of attachment for the chloroplasts. The latter characteristic was valid also for the symbionts isolated from Haplodiscus sp. and Amphiscolops sp. (Trench & Winsor,

54 1987; Lopes & Silveira, 1994). In our study, no evidence of radiating chloroplasts was

found. The multiple and condensed chromosomes observed in the larger symbiont of

Waminoa sp. resemble those described in Amphidinium sp. from Haplodiscus sp. (Trench

& Winsor, 1987), but differ from that described for Amphidinium klebsii in Amphiscolops langehansi (Taylor, 1971). The presence of a flagellum in hospite, and the general size and shape lead us to propose that the larger symbiont is an Amphidinium.

Algal symbionts within the worm were each surrounded by cytoplasmic processes originating from the worm’s parenchyme cells and found in close proximity to their nuclei, thus implying a possible intracellular position of the symbionts. Coexistence of two species of algal symbionts was previously described in species of the convolutid

Amphiscolops (Yamasu & Okazaki, 1987), in Waminoa litus (Winsor, 1990) and in

Haplodiscus sp., where the two co-occurred within the same host cell (Trench & Winsor,

1987). Trench & Winsor (1987) speculated that the two symbiont types might be involved in a competitive exclusion process, yielding only one algal type in adult worms.

Since sexually mature specimens of Haplodiscus sp. were lacking, it was impossible for the authors to confirm this assumption. In our study, sexually mature specimens contained both symbiont types, thus negating the occurrence of competitive exclusion.

An additional speculation made by Trench & Winsor (1987) was that the Amphidinium was the natural symbiont and the Symbiodinium was acquired from the corals on which the worms live. However, they specifically mention that there is no evidence that the worms fed on any of the host corals (Trench & Winsor, 1987).

55 To study the physical interaction between corals and worms we used two approaches.

The first one checked whether the worms damage the coral and compared the morphology of the surface of corals that were infested with worms with that of corals devoid of worms. The second one studied the identity of Symbiodinium sp. symbionts in both corals and worms and examined the genotype of Symbiodinium sp. in the two partners using PCR-DGGE (LaJeunesse, 2001).

The SEM images of the surface of the coral Stereonephthya cundabiluensis with and without worms (Fig. 4.5) suggest that the worms may be removing the surface mucus layer, using their dense cilia cover to brush off the mucus. Possible nutritional value of the mucus (see Wild et al., 2004) for the worms awaits further examination. Although these SEM images indicated that the tissue of infested colonies did not contain any lesions or injuries there is a possibility that the worms interfere with certain biochemical processes of the coral and its algal symbionts, such as photosynthesis, by causing a

“shading” effect when present in high numbers on its surface. Furthermore, a recent review by Brown & Bythell (2005) highlights a variety of functions of coral mucus layer mostly as a defending agent against: pathogens, space invasion by other corals, UVR damage, desiccation, smothering by sediment and against pollutants. Considering these data, the removal of the mucus by the worms might make the coral more susceptible to various biotic and abiotic disturbances.

The Symbidinium sp. symbionts in the worm often differed genetically to those in the

"host" coral colony (Fig. 4.6). This marked difference in symbiont complement implies that the worm acquires its symbionts elsewhere. To this end we have determined that the worms vertically transmit both Amphidinium sp. and Symbiodinium sp. to the oocytes

56 during gametogenesis (see Section V). Therefore, the juvenile worm inherits symbionts

from the parent and may maintain them through to reproductive maturity. Predation on a

cnidarian host and incorporation of zooxanthellae into the predator’s tissue has been

recorded in several nudibranch snails such as Spurilla neapolitana (Marin & Ros, 1991).

Such “second hand” zooxanthellae are known to be acquired via feeding and translocated

to vacuoles in the cytoplasm of endothelial cells of the digestive gland (Kempf, 1991;

Marin & Ros, 1991). Based on consistent differences in symbiont identity between the

worms and their coral hosts as was shown in the DGGE analysis (Fig. 4.6), and the lack

of any noticeable tissue loss on "host" corals, it is concluded that the worms do not prey

on their coral hosts, nor do they acquire their algal symbionts directly from them.

The PCR-DGGE fingerprints of algal symbionts derived from 6 colonies of Turbinaria

sp. corals and their worms revealed high consistency in the identity of symbionts found in both the worms and their coral hosts. This consistency might indicate that the worms which occupy a single coral colony are relatively stationary and form a uniform group in terms of algal symbiont content. However, there were two groups of worms (isolated from the corals Turbinaria sp. and Stylophora pistillata) which did show heterogeneity in

algal symbiont content (possessing algae belonging to two different sub-clades), a finding that may indicate that a batch of worms residing on one coral colony could contain worms differing in their Symbiodinium genotype. At this stage, when the number of species of Waminoa in Eilat is not known as well as whether different species of worms contain different types of algal symbionts it is hard to generalize regarding this finding.

As for the possibility that the worms simultaneously contain two different genotypes of

Symbiodinium, it is suggested that this scenario is less likely and that the result obtained

57 is an artifact that may have resulted from the use of a large pool (30-50) of worms derived from one coral colony for the DNA extraction (several worms in the batch contained Symbiodinium symbionts belonging to a different sub-clade than the rest).

The occurrence of acoelomorph worms on many coral hosts in Eilat reefs may be a unique phenomenon. The extent and prevalence of epizoic symbiotic acoelomorph worms on corals in other regions around the Indo-Pacific is virtually unknown. This interaction comprised of two invertebrate hosts harboring algal symbionts and living together exemplifies the complexity and interdependence of life on a coral reef.

Determining the relative energetic contribution each of these associations provides, would further elucidate their importance.

58 SECTION V

Sexual and asexual reproduction and mode of symbiont

acquisition in Waminoa brickneri

5.1 Introduction

Acoelomorphs are hermaphroditic (Brusca & Brusca, 1990) members of the

Archoöphora, a group within the platyhelminthes which include the Nemertodermatida,

Macrostomida and Polycladida characterized by homocellular female gonads consisting

of entholecithal eggs and devoid of vitelline cells (Raikova et al., 1995). Sexual

reproduction and oogenesis have been studied mostly in the neoophoran flatworms and to

much less extent in archoophorans (Falleni & Gremigni, 1990; Raikova et al., 1995). The

general reproductive strategy of the vast majority of turbellarians* is to produce relatively

few zygotes, which are protected by brooding or encapsulation and undergo direct

development (Henley, 1974; Brusca & Brusca, 1990). All of the symbiotic flatworms

studied to date produce aposymbiotic eggs (McCoy & Balzer, 2002). To date, there is no

data available concerning the mode of sexual reproduction in the genus Waminoa.

As with many other animal groups, classification of the acoel species is based on the variety of form of their reproductive organs (Tyler, 2003). The ultrastructural features of spermatozoa of Acoelomorpha are a rich source of distinctive characters which usually agrees with molecular systematics of this group (Petrov et al., 2004).

Horizontal transmission of algal symbionts in the Acoelomorpha is well documented

(Douglas, 1992). The sexual reproduction, egg production and the uptake of algal

59 symbionts by the juveniles of Symsagittifera roscoffensis was extensively studied and

became a model for specificity studies in this group (Douglas & Gooday, 1982; Douglas,

1992). The acoel Amphiscolops carvalhoi, however, was described as having

zooxanthellae in internally brooded young (Marcus, 1954), implying possible maternal

transmission. Without information on the presence of algal symbionts in its oocytes,

however, evidence of vertical transmission is lacking.

Waminoa brickneri, a newly discovered species from the reefs of Eilat (Red Sea) is

epizoic on living corals (Section IV). Similar worms belonging to the genus Waminoa

were detected on 14 species of stony and soft corals at a depth range of 2-50 meters

(Section IV). The worms possess two distinct dinoflagellate algal symbionts within their cells: small symbionts 5-10 µm in diameter, which were identified as belonging to the

genus Symbiodinium (Section IV) and larger yet-to-be identified symbionts 12-20 µm in

diameter. The initial hypothesis that the worms receive their algal symbionts from their

coral hosts was examined using Denaturing Gradient Gel Electrophoresis (DGGE)

profiles of the ITS2 region of Symbiodinium derived from coral hosts and resident worms

(see Section IV). However, it was determined that corals and worms possess different

phylotypes of Symbiodinium, thus suggesting a different source for their symbionts.

In this part of the work I studied sexual and asexual reproduction in the acoel Waminoa

brickneri and determined the mode of algal acquisition for this species.

5.2 Materials and Methods

5.2.1 Collection and maintenance of animals: A part of a colony of Plesiastrea laxa with epizoic worms was collected near the Inter University institute in Eilat (Red Sea) on

60 31.3.04 and transferred to an aerated aquarium at Tel Aviv University. The worms were

removed from their coral host and kept in Pyrex bowls containing 250 ml of 45 μm filtered sea-water. 20 worms were preserved in 2.5% glutaraldehyde in sea-water on the day of collection. Observations on the live worms (~150 worms in each bowl) were carried out daily with the aid of a dissecting microscope.

5.2.2 Histology: Worms were preserved in 2.5% glutaraldehyde in sea-water and put into

2% liquid agarose gel. After the gel solidified it was dehydrated in a graded series of ethanol and butanol and embedded in paraffin. Serial sections 8 μm thick were cut using

MIR microtom (Shandon). Sections were stained with Delafield Hematoxylin-Eosin.

Specimens were preserved on March 31st (the day of collection), on April 14th and April

19th (a day after the egg masses were detected).

5.2.3 Transmission electron microscopy of worms: Worms were preserved in 2.5%

glutaraldehyde in sea-water, rinsed in buffer phosphate, stained with 1% OsO4, dehydrated through a graded ethanol series and embedded in Epon. Sections were cut with a diamond knife, stained with lead citrate, and viewed with JEOL 1200 EX transmission electron microscope.

5.2.4 Asexual reproduction of worms

Twenty worms from each of the corals P. laxa, A. hemprichi and S. cundabiluensis were removed and transferred to 12-well tissue culture plates. Each well contained one worm in 3 ml 0.45 μm filtered sea-water (FSW). Half of the water volume was changed every second day. The plates were maintained in an incubator (Yihdern, LE-509), at a temperature corresponding to the ambient seawater temperature at time of collection. The light regime was 12h light (30 μmol quanta m-2 s-1): 12h dark. Survivorship and asexual

reproduction of the worms were monitored every three days with the aid of a dissecting

61 microscope. The percentage of worms that underwent asexual reproduction was

calculated. Experiments were preformed during December 2003 - January 2004 and

February - March 2004.

5.3 Results

I studied the sexual reproduction of Waminoa brickneri removed from the stony coral

Plesiastrea laxa (Fig. 5.1A). The documentation of events started upon the collection of

the coral with the worms, which was 18 days prior to egg lying, and lasted until hatching

of the juveniles, which occurred 4 days afterwards. Sexually mature worms, containing

paired ovaries and testes, were observed on the day of collection. TEM images of these

worms show a male gonad with sperm in close proximity to a female gonad containing

oocytes with prominent nuclei and nucleoli. Sperm cells are elongated (Fig 5.2A) and biflagellate (Fig. 5.2B). The axonemes of both flagellas have a 9+0 arrangement. Fig.

5.2C shows an algal symbiont engulfed by cytoplasmic processes originating from an

oocyte. Sixteen days after collection, histological sections of other specimens from the

same batch of worms revealed the presence of few elongated oocytes within the ovary.

These were occasionally stretched dorso-ventrally (Fig. 5.1C). Large nuclei with distinct nucleoli were detected (Fig.5.1D). Algal symbionts were observed within the ooplasm of developing oocytes (Fig. 5.1D). Fig. 5.1E shows an oocyte with 2 distinct types of algal

symbionts (see Introduction) with the smaller one being more abundant. 3 Gelatinous egg

masses were found eighteen days after collection, attached to the bottom of the bowl,

each containing 2-9 eggs (Fig. 5.1F). The eggs were each 200 μm in diameter and brown

in color due to the presence of algal symbionts. A transparent capsule, 0.8 μm thick,

62 coated each egg. TEM micrographs of the embryo within the egg capsule revealed a

ciliated epidermis, numerous nuclei of parenchymal cells and algal symbionts within the

parenchyma (Fig. 5.3A). Lipid droplets, attached to the inner face of the egg capsule and

project to the gap between the embryo’s epidermis and the capsule were detected (Fig.

5.3B). Three days after eggs were laid, embryos were observed moving within the egg

capsule. At the fourth day after eggs were laid, while watching the eggs under a

dissecting microscope, a worm hatched from one of the eggs. The hatchling, 190 μm in

diameter, containing a statocyst, two eyespots and abundant algal symbionts was actively

swimming in the water (Fig. 5.1G). A total of four eggs hatched, while the rest (a batch

of 2 and a batch of 9) were preserved for electron microscopy.

Worms isolated from P. laxa underwent asexual reproduction in the laboratory at a

rate of 40-45%, in S. cundabiluensis at 30-35%, and in A. hemprichii at 5-10% over the

course of 33 days (n=40 worms from each coral host). During the process, the worms

released fragments of biomass of various sizes, from their body. On a few occasions the fragments were as small as 350-500 μm in diameter and resembled tiny spheres, which I

termed "worm balls" (Fig. 5.4). These were seen to be actively moving, covered with

beating cilia, as later confirmed by observation through a dissecting microscope.

63

Figure 5.1 (A) The stony coral Plesiastrea laxa with Waminoa brickneri worms. Scale bar 3 mm. (B-F) Stages of sexual reproduction in W. brickneri. (B) Ventral view of a sexually mature specimen with paired ovary (ov) and the male copulatory apparatus (mca) circled with dashed line. Scale bar = 1 mm. (C) Histological section of worm containing gonads, 5 days prior egg laying, showing elongate oocytes. Scale bar = 110 μm. (D) Oocyte containing nucleus (n) with prominent nucleolus and algal symbionts (arrows). Scale bar = 30 μm. (E) Oocyte containing two symbionts types: Symbiodinium sp. (black arrow) and single larger symbiont (white arrow). Scale bar = 30 μm. (F) Gelatinous egg mass. Scale bar = 200 μm. (G) Worm-hatchling containing numerous algal symbionts, statocyst (black arrow) and eyespot (white arrow). Scale bar = 45 μm. 64 Figure 5.2 Waminoa brickneri TEM micrographs of gonads: 18 days prior to egg laying. (A) Ovary containing oocytes with prominent nuclei (n) and nucleolus (black arrow) in close proximity to testes containing sperm (s). Scale bar = 2 μm. (B) Cross section of biflagellate sperm cells characterized by 9+0 axoneme arrangement. Scale bar = 200 nm. (C) Zooxanthella (z) engulfed by cytoplasmic processes (arrows) originating from adjacent oocyte (o). Scale bar = 2 μm.

65

Figure 5.3 Waminoa brickneri TEM micrographs of: embryo within egg capsule. (A) Egg capsule (black arrows), ciliated (asterisks) portion of an embryo containing algal symbionts (z) and nuclei (n) of parenchyme cells. Scale bar = 5 μm. (B) Inner face of egg capsule (ec), sections through epidermal cilia in gap between embryo and egg capsule and electron-dense body (asterisk). Scale bar = 500 nm.

Figure 5.4 Asexual reproduction in Waminoa sp. An adult worm and a “worm ball”. Scale bar 1 mm.

66

5.4 Discussion

This study represents the first documentation of sexual reproduction in the symbiotic acoelomorph genus Waminoa. Furthermore, it provides the first evidence of maternal transmission of algal symbionts at the oocyte stage in any triploblastic organism studied to date. It also suggests that this symbiosis represents a closed system where the host retains its symbionts from generation to generation although there is a possibility for potential uptake of additional symbionts from the environment. To date, all the host flatworms studied are known to produce aposymbiotic eggs within a mucilaginous egg case (McCoy & Balzer, 2002). Potential symbionts, and other algae, have been found on the egg cases of several host species (Douglas & Gooday, 1982; McCoy & Balzer, 2002), but never inside them.

The presence of two symbiont types within the oocytes of Waminoa brickneri from Eilat is the first report of the vertical transmission of two distinct algal symbionts. Our finding, regarding vertical transmission of both symbionts types in Waminoa sp. from Eilat demonstrates that the juvenile worms are equipped with a pool of symbionts from a maternal source upon hatching, without a need to rely on any external sources. Hence, the habitat range of Waminoa brickneri does not seem to be constrained by the distribution of free-living populations of suitable symbionts as known from systems with horizontal transmission (Douglas, 1992). Although it could be constrained by environmental factors needed to support the symbiosis such as light and nutrients availability.

The basal position of the Acoelomorpha and the consequent polyphyly of the

Platyhelminthes are continuously being tested using both morphological and molecular

67 techniques (Ruiz-Trillo et al., 2004). Analyses of mitochondrial amino acid sequences

concurrent with morphological characters and a growing number of molecular evidences

argue for a monophyletic Acoelomorpha as a basal bilaterian group, occupying a pivotal

position between diploblast and triploblasts (Ruiz-Trillo et al., 2004). The finding

concerning the maternal transmission of algal symbionts in this group can be regarded as

a significant trait which further strengthens the new position of the Acoelomorpha as the

most basal known triploblastic Bilateria (Baguna & Riutort, 2004; Glenner et al., 2004).

Such mode of symbiont transmission has never been documented in the Platyhelminthes

(McCoy & Balzer, 2002), however, is well known from the diploblastic cnidarians

(Douglas, 1994).

Waminoa sp. Worms were reported to associate with 14 species of stony and soft corals

in Eilat (Section IV). Sexually mature specimens were scarce and the event of sexual

reproduction in laboratory-raised specimens during the month of April is regarded as exceptional. Further attempts to re-document the sexual reproduction scenario in following months were unsuccessful. It is known that the development and maintenance of the female reproductive system in members of the Turbellaria can be affected by exogenous factors, among which seasonal factors and particularly temperature may cause the alteration between sexual and asexual states (Ax, 1977). We hypothesize that asexual reproduction is the common mode of reproduction in Waminoa sp. From Eilat while sexual reproduction might be seasonal.

The sperm of Waminoa brickneri appears elongated and biflagellate. A cross section through both flagella revealed a 9+0 arrangement of the axonemes. Such an arrangement

68 is typical to “large-bodied convolutids” (see Petrov et al., 2004) and interestingly also to large-bodied acoels containing algal endosymbionts (Hooge et al., 2002). Recently

Ogunlana et al., (2005) suggested re-assigning Waminoa to the Convolutidae based on morphological and molecular features. The sperm morphology, as seen in this study further corroborates this assignment.

My study provides the first evidence for asexual reproduction in the genus Waminoa, which matches the characteristics of architomy (Åkesson et al., 2001). This process involves the separation of fragments or whole body parts from the mother animal prior to organ differentiation. A similar type of architomy was described in the acoelomorph

Amphiscolops langerhansi, which was reported to release odd-shaped little worms

(Hanson, 1960). The exact cascade of events involved in this process in Waminoa sp. and the seasonal rate of this mode of reproduction is yet to be determined.

The symbiosis between Waminoa brickneri and its endosymbionts and its intimacy with another symbiotic system involving corals and algal symbionts is intriguing and complex.

The data obtained in this section of the study concurrent with a morphological study of

Waminoa sp. and algal symbionts and a molecular account of Symbiodinium symbionts from worms and coral hosts (see Section IV) provide insight into this intricate symbiosis system, which was so far unexplored. The results of this study undoubtedly will intrigue future research needed for the elucidation of key features especially related to the coral- worm relationship. So far, the only finding, which hints on the interaction between worms and the coral, is the removal of mucus by the worms, but still, other important aspects such as nutrition on mucus by the worms, nutrients trade between coral and

69 worms and possibly competition for light by the two algae-containing players (coral and worms) remains to be explored.

70 SECTION VI

Summary and Conclusions

The study of symbiotic systems frequently demands a multi-disciplinary approach. Given

the fact that such systems are comprised of at least two partners, one needs to relate to each of the partners according to his/hers research questions, employing various techniques. My study focuses on the symbiotic relationship involving corals and

unicellular algae as well as acoel worms harboring such algae and found epizoic on

corals. In this research I studied the initiation of the symbiotic relationship between the

soft coral Heteroxenia fuscescens and its algal symbionts in search for symbiosis-related

proteins in the coral host, I looked for specificity of algal symbionts and soft coral hosts

in relation to the coral mode of symbiont acquisition, and studied a newly discovered

symbiotic relationship involving acoel worms, corals and symbiotic algae.

The first part of my work (Section II) concentrated on the coral host and its first

encounter with symbiotic algae. In this part I tried to unravel molecular changes

occurring in the juvenile coral host at different time intervals (days and weeks) after the

acquisition of symbiotic algae. This is the first work to search for symbiosis-specific

proteins during the natural onset of the symbiosis in early host ontogeny. As a tool, I used

2D PAGE technique to compare aposymbiotic and symbiotic primary polyps of the soft

coral Heteroxenia fuscescens. The results of the comparative proteomic study revealed

changes in the host soft coral proteome through development, but surprisingly virtually

no changes in the host proteome as a function of symbiotic state. This suggests that

during the first days and weeks of symbiosis between Heteroxenia fuscescens and

71 Symbiodinium sp., translational and post translational changes specific to the symbiotic state may not be occurring in the host. These finding are remarkably similar to a recent

comparative study of symbiotic and aposymbiotic larvae of the stony coral Fungia

scutaria (deBoer, 2004; deBoer & Weis, in preparation). Two dimensional PAGE

patterns of host proteins revealed only one consistent difference out of approximately 450

proteins between symbiotic and aposymbiotic larvae of the same age.

There are several possible explanations for the uniformity of proteome patterns between

symbiotic and aposymbiotic hosts. It is possible that changes are occurring but they are

not being detected. This could be due to a variety of factors including slow protein

turnover with age of the host proteome, transient expression of symbiosis-specific or

enhanced proteins, and expression of these proteins in very low quantities below

detection levels. If symbiosis-specific protein expression is indeed limited to those cells

housing the symbionts, then these host cells number only in the hundreds to thousands in

the early weeks of the symbiosis. Yacobovitch (2001) followed the dynamics of algal

acquisition in laboratory reared and naturally settled primary polyps of H. fuscescens and

showed a gradual increase of algae uptake starting at 90 algal cells per polyp at seven-

day-old polyps increasing to 15000 at 56-day-old ones. In a background of many

thousands of cells comprising a juvenile polyp, any symbiosis-specific protein signal

might be lost. An example that further demonstrates this possibility is in E. scolopes,

where a differential result was obtained only in its symbiosis-specific organs that had

been dissected away from the rest of the animal (Doino Lemus & McFall-Ngai, 2000).

Since in Cnidaria algal symbionts are not housed in such a specific location, detecting

these symbiosis-specific changes is made difficult by a high non-symbiosis cell

background. Another potential explanation could be related to the methodology used for

72 the isolation of algal symbionts prior the procedure of protein extraction through centrifugation. If indeed the symbiosis-specific protein expression is limited to the cells housing the symbionts or to the membranes surrounding them, there is a possibility that part of these membranes are staying attached to the symbionts and are being tossed along with their potential symbiosis-related proteins. In conclusion, the onset of symbiosis between H. fuscescens and Symbiodinium sp. seems not to be accompanied by pronounced changes in the host proteome in the first weeks of the association. Further studies employing more sensitive techniques, such as cDNA microarrays, are needed to identify proteins involved in the initial interactions between the partners.

The second part of my study (Section III) examined the algal symbiont identity in multiple coral host species in one location and provides, for the first time, perspective on the mode of symbiont acquisition and its relation to clade specificity in a variety of soft coral hosts. It was discovered that all species harboring clade C algae acquire their symbionts horizontally from the environment, while the species harboring clade A algae acquire theirs’ directly from the parent at the oocyte stage.

The limitation of clade A symbionts to hosts with vertical transmission suggests a coevolution of the hosts and symbionts. In certain symbiotic systems such as aphid- bacteria symbiosis (Douglas, 1994) it has been discovered that the phylogeny of the aphid symbionts parallels their hosts because the bacteria are vertically transferred. It has been suggested that the association probably evolved in the common ancestor of the

Aphidoidea and the bacteria have been passed from mother to offspring. As the

73 aphidoidea diversified, each aphid lineage carried its own complement bacteria with it

(Douglas, 1994). Data on the phylogeny of Red Sea soft corals that could be overlaid

onto the phylogeny of the symbionts will yield further information on how the observed

specificity pattern arose. Were clade A algae “captured” by a single nephtheid ancestor

that gave rise to the three vertically transmitting genera, or did this event occured

multiple times within the family?

Clade C symbionts, characterized by large sub-clade variability, are found in corals with

horizontal transmission and, most probably, each of its genotypes exhibits a more specialized set of physiological capabilities. LaJeunesse et al. (2003) found that the majority of endosymbiotic dinoflagellates in cnidarians in the GBR (Austarlia) belong to

Clade C, which is composed of closely related, yet ecologically and physiologically

distinct types. The observed combination of two different genotypes of clade C in 5

species of alcyoniid soft corals further highlights the flexibility of an open system of

symbiont acquisition. To date, no studies have systematically investigated the diversity

of free-living Symbiodinium in the environment (Baker, 2003). Several studies have

identified apparently free-living Symbiodonium in the waters or sediments surrounding

potential invertebrate hosts (Taylor, 1973, Loeblich & Sherley, 1979, Carlos et al., 1999).

In a study aimed at comparing the survivorship, development and symbiont acquisition of

laboratory vs. field reared primary polyps of the soft coral Heteroxenia fuscescens in

Eilat, the planulae were placed in closed PVC containers (each container had 4 pieces of

plankton net on its 4 walls and a plastic lid on top) containing granite gravel, which were

anchored to the reef (Yacobovitch et al., 2003). These polyps became naturally infected by zooxanthellae and had no contact with the reef sediment, nor possible contact with

74 predators’ feces (Yacobovitch et al., 2003). Hence, it is suggested that the symbionts were derived from the water column. Thus, we hypothesize that free-living stages of clade C symbionts are prevalent in the reef area and are available for acquisition by juvenile stages of soft coral hosts. Future studies examining distribution and availability of symbionts in the open water and physiological plasticity of different symbionts strains are needed to test these hypotheses.

Based on our results in soft corals and on the recent literature concerning the stony corals

Montipora and Porites (LaJeunesse et al, 2003, Van Oppen, 2004,), we hypothesize that corals, which vertically transmit their symbionts, will tend to have genetically distinct symbionts, which differ from symbionts found in corals with horizontal transmission. In our study the distinctive feature is symbiont clade, while in Montipora and Porites the symbionts were found to belong to a distinct sub-clade. Whether the symbionts belonging to clade A (from the three studied nephtheid corals) represent one type or 3 similar types, is yet to be determined.

Vertical transmission is relatively rare among cnidarians (Trench, 1987). Coral hosts with vertical transmission of symbionts are assumed to represent a scenario in which a certain ancestral symbiont was “trapped” and evolved within host tissues. It is likely that these holobionts were exposed to changing environmental conditions during their evolution and therefore, their survival indicates their resilience. Several questions have emerged regarding corals which vertically transmit their symbionts: Is it possible that such corals are more resistant to environmental change? Are such coral less susceptible to bleaching?

We are only at the beginning of understanding these issues however, there are some data

75 that may support a positive answer to these questions. Among a variety of GBR stony corals, two of the most bleaching-resistant species were Montipora digitata and Porites cylindrica, which both have vertical transmission of symbionts and associate with the unique Symbiodinium strain C15 (LaJeunesse et al., 2003). A comparative approach of sampling symbionts from hosts with vertical vs. horizontal transmission and analysis of their physiological capabilities can answer these important questions.

The results obtained in this part of the study and the questions raised above, led me to reflect on the basics of the important hypothesis suggested by Buddemeier and Fautin

(1993) termed The Adaptive Bleaching Hypothesis (ABH). The ABH posits that when environmental circumstances change, the loss of one or more types of zooxanthellae is rapidly, sometimes unnoticeably, followed by formation of a new symbiotic consortium with different zooxanthellae that are more suited to the new conditions in the host’s habitat. One of the fundamental assumptions of the ABH is that bleached adults can secondarily acquire zooxanthellae from the environment (Kinzie et al., 2001). This assumption is well tested for anthozoans with horizontal transmission (Schoenberg and

Trench, 1980; Davy et al., 1997; Toller et al., 2001; Kinzie et al., 2001), however evidence is still lacking for the potential of species with vertical transmission to re- acquire zooxanthellae from the environment. This important piece of information will probably be elucidated with the availability of molecular markers that can trace accurately different types of zooxanthellae regardless of their quantity inside the host. If corals with vertical transmission are indeed closed systems it will be interesting to reveal what are the mechanisms which enable them to cope with acute changes in environmental settings.

76

The third and fourth parts of my work (Sections IV and V) concentrated on the symbiotic

relationship involving acoel worms harboring dinoflagellate algae and corals on which

these worms reside. This study is the first to look into aspects of the interaction between the worms and their coral host as well as the reproduction and acquisition mode of the symbiotic algae by these worms. The acoelomorph worms on Eilat's coral reefs were identified as belonging to the genus Waminoa, which was previously described from

Australia (Winsor, 1990). Sexually mature worms from the coral Plesiastrea laxa were

identified as a new species named Waminoa brickneri (Ogunlana et al., 2005). Prior to

our study, the only other described species in this genus is W. litus (Winsor, 1990).

Worms isolated from the stony corals Acropora hemprichi, Stylophora pistillata and

Plesiastrea laxa exhibited variability in morphological features, such as size and

reproductive state, which might be indicative for the presence of several species of

Waminoa in Eilat. The classification of acoelomorph species is based on characteristics

of their reproductive organs (Tyler, 2003).

The lamellose coral species Turbinaria sp. and Echinophyllia sp. showed the highest

infestation ratio (Table 4.1). However, it should be noted that in these two taxa only one

colony out of five was infected. On the other hand, in the branching coral S. pistillata,

which is one of the most abundant stony corals in Eilat (Loya, 1972), only five out of 225

sampled colonies were infected. Thus, infestation rate in the study area appears to be low

and patchy in nature.

Only worms from A. hemprichi contained genital openings. A low occurrence of sexually

mature acoelomorph worms has been also encountered in other studies of Waminoa sp.

77 and Haplodiscus (Trench & Winsor, 1987; Winsor, 1990). Such findings suggest that

sexual reproduction might be seasonal.

The worms contained two distinct algal symbionts. The smaller ones, which possessed a double-stalked pyrenoid, were assessed molecularly using PCR-DGGE of the ITS-2 region (LaJeunesse, 2001) and were found to belong to the genus Symbiodinium. The larger symbionts were characterized by the presence of multiple chloroplasts, an irregular cell margin and the presence of a flagellum and are presumed to belong to the genus

Amphidinium.

The algal symbionts observed within the worms were each surrounded by cytoplasmic processes originating from their parenchyme cells and found in close proximity to their nuclei, thus implying a possible intracellular position of the symbionts. Coexistence of two species of algal symbionts was previously described in species of the convolutid

Amphiscolops (see: Yamasu & Okazaki, 1987), in Waminoa litus (see: Winsor, 1990) and

in Haplodiscus sp., where the two co-occurred within the same host cell (Trench &

Winsor, 1987). Trench & Winsor (1987) speculated that the Amphidinium might be the

natural symbiont and that the Symbiodinium was acquired from the corals on which the

worms live. However, they specifically mention that there is no evidence that the worms

fed on any of the host corals (Trench & Winsor, 1987).

In order to study aspects of the physical interaction between corals and worms we used

two following approaches: (1) in order to check whether the worms injure the coral we

conducted a comparative morphological examination of the surface area of corals that

78 were infected with worms vs. corals that were devoid of worms and (2) in order to check

the identity of Symbiodinium sp. symbionts in corals and worms we examined the

genotype of Symbiodinium sp. in corals and their resident worms using PCR-DGGE

(LaJeunesse, 2001). The comparative images of the surface area of the coral

Stereonephthya cundabiluensis with and without worms suggest that the worms might be

involved in removal of the coral surface mucus layer, using their dense cilia cover to

brush off the mucus from the surface. Possible nutritional value of the mucus (see Wild et

al., 2004) for the worms awaits further examination. The SEM images indicated that the

tissue of infected coral colonies did not contain any lesions or injuries. However, there is

a possibility that the worms interfere with certain biochemical processes of the coral such

as photosynthesis, by causing a “shading” effect when present in high numbers. The molecular work strengthens my morphological results since I discovered that the

Symbiodinium sp. genotype in coral hosts and their resident worms differs, thus, negating our working hypothesis. Moreover, we discovered that the worms vertically transmit both symbiont types at the oocyte stage (Section V). The latter finding demonstrates that the juvenile worm is equipped with a pool of symbionts upon hatching and therefore, not

obligated to acquire symbionts from an external source (such as its coral host). Predation

on a cnidarian host and incorporation of zooxanthellae into the predator’s tissue is known

from several species of nudibranchs. Such “second hand” zooxanthallea are known to be

acquired via feeding and translocated to vacuoles in the cytoplasm of endothelial cells of

the digestive gland (Marin & Ros, 1991). Based on my results I conclude that the worms

do not prey on their coral hosts nor do they acquire their algal symbionts directly from them.

79 The PCR-DGGE fingerprints of algal symbionts derived from 6 colonies of Turbinaria

sp. Corals and their worms revealed high consistency in identity of symbionts found in

worms as well as coral hosts. This consistency, might indicate that the worms which

occupy a single coral colony are relatively stationary and form a uniform group in terms

of algal symbionts content, however there were 2 groups of worms which did show

heterogeneity in algal symbionts content (posses 2 different sub-claeds), therefore this

shows that a batch of worms residing on one coral colony could contain worms differing

in their Symbiodinium genotype.

The documentation of sexual reproduction in Waminoa brickneri provided the first evidence of maternal transmission of algal symbionts at the oocyte stage in any triploblastic organism studied so far. To date, all host flatworms studied are known to produce aposymbiotic eggs within a mucilaginous egg case (McCoy & Balzer, 2002).

Potential symbionts, and other algae, have been found on the egg cases of several host species (see: McCoy & Balzer, 2002), but never inside them. Furthermore, this is the first known occurrence of simultaneous vertical transmission of two distinct algal symbiont species.

The basal position of the Acoelomorpha and the consequent polyphyly of the

Platyhelminthes are being tested using both morphological and molecular techniques

(Ruiz-Trillo et al., 2004; Glenner et al., 2004). Analyses of mitochondrial amino acid sequences concurrent with morphological characters and a growing number of molecular studies argue for a monophyletic Acoelomorpha as a basal bilaterian group, occupying a pivotal position between diploblast and triploblasts (Ruiz-Trillo et al., 1999). The 80 maternal transmission of algal symbionts in W. brickneri is the first recorded among

triploblastic Bilateria. Such mode of transmission has never been documented in the

Platyhelminthes (McCoy & Balzer, 2002), however, is well known from the diploblastic

cnidarians (Douglas, 1994).

This part of the study documents for the first time the occurrence of acoelomorph worms,

belonging to the genus Waminoa, on several coral hosts in Eilat reefs. This symbiotic

system, comprised of two invertebrate hosts harboring algal symbionts and living together exemplifies the complexity and interdependence of life on a coral reef. The extent and prevalence of epizoic symbiotic acoelomorph worms on corals in other regions around the Indo-Pacific is virtually unknown. The presence of the worms strictly on live corals is to my opinion not a coincidence. To date, the only finding in support of this assumption is the absence of a mucus sheet in corals harboring the worms. Future studies examining possible nutrients and metabolites transfer between the worms and corals and determining the relative energetic contribution each of these associations provides will supply valuable information regarding the nature of this presumed three party symbiosis

There are several other questions which remain to be answered:

1. How do the worms avoid predation while crawling on the coral surface?

2. How do the worms live undisturbed on the living tissue of the coral?

3. How is the photosynthesis process of the coral affected by the presence of

the worms?

4. What is the mode of dispersal of the worms from one coral host to the

other?

5. How many species of worms are found in Eilat? 81

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אורית ברנע היבטים מולקולריים וספציפיות במערכות סימביוזה ימיות הכוללות אצות חד-תאיות

תקציר קיומן של שוניות אלמוגים במשך מיליוני שנים במים דלי נוטריינטים, המתאפיינות בכך, שאור השמש חודר למעמקיהם, קשור ללא ספק לנוכחותן של אצות חד-תאיות, המצויות ברקמות אלמוגים. הסימביוזה בין אורגניזמים, השייכים למערכת הצורבים ובין אצות חד תאיות, נחשבת כאחת החשובות בסביבה הימית ויש לה תפקיד קריטי במארג המזון ובנית התשתית של שונית האלמוגים. מערכות סימביוזה המושתתות על יחסי גומלין בין שתי ישויות שונות, תמיד שימשו בסיס למחקרים, העוסקים בספציפיות בין המארח והסימביונט. בהקשר לסימביוזה צורבים-אצות, נושא הספציפיות מהווה אגוז קשה לפיצוח, דבר הנובע מכמה סיבות: (1) הטקסונומיה של האצות השיתופיות עדיין לא ברורה והינה בשלבי למידה, 2( ) קיימים שני מנגנונים של רכישת אצות בקרב אורגניזמים ממערכת הצורבים, 3( ) קיימים מאות מינים של מארחים ומספר לא ידוע של מיני סימביונטים. גורמים ביוטיים וא-ביוטיים רבים ומגוונים כמו עומק, תאורה, ביוגיאוגרפיה, והתפתחות אונתוגנטית נלמדו בהקשר לשאלת הספציפיות של היחסים בין שני השותפים. גורם חשוב נוסף המעורר עניין, הוא מנגנון רכישת האצות על ידי האורגניזם המארח. סימביונטים יכולים להיות מועברים ישירות מההורה לצאצא המיני במנגנון הקרוי העברה ורטיקלית. במנגנון זה, מועברים הסימביונטים מגוף ההורה ישירות לתאי הביצה, המתפתחים בו. מנגנון נוסף, קרוי העברה הוריזונטלית ובו נרכשים הסימביונטים על ידי דרגה התפתחותית צעירה מהסביבה החיצונית. העברה ורטיקלית של אצות שיתופיות נדירה יחסית בקרב אורגניזמים ימיים ותוארה במינים ממערכת הספוגים ובמינים ממערכת הצורבים. בעוד שבמערכת הסימביוזה צורבים-אצות ידועים שני המנגנונים שצוינו לעיל, בתולעים השייכות למערכה, שהוגדרה רק לאחרונה Acoelomorpha ידועה עד היום רק העברה הוריזונטלית. תהליך יצירה של סימביוזה מלווה לרוב בשינויים מורפולוגיים, פיזיולוגיים, ביוכימיים ומולקולריים בשני השותפים למערכת. בסימביוזה בין אלמוגים ואצות, המנגנונים הביוכימיים והמולקולריים , המאפיינים , את שלב יצירת הקשר בין שני השותפים, עדיין לא נלמדו. מערכת הסימביוזה צורבים-אצות נחקרת רבות בעשורים האחרונים ומושכת אליה את רוב תשומת הלב בעיקר עקב תופעת ההלבנה. בניגוד לצורבים, מיני תולעים השייכים למערכות Platyhelminthes ו - Acoelomorpha, החיים בסימביוזה עם אצות שיתופיות, כמעט ולא נלמדו. באילת נצפו תולעים ממערכת ה - - Acoelomorpha כשהן חיות על אלמוגים. הנוכחות של אצות שיתופיות, הן באלמוגים והן בתולעים החיות עליהן והקשר הפיזי הקרוב ביניהם (תולעים ואלמוגים), עוררה שאלות רבות לגבי אופייה של מערכת סימביוזה ייחודית זו.

1 המחקר הנוכחי נערך בשונית האלמוגים של אילת ובו נלמדו היבטים שונים כמו ספציפיות, מגוון ביולוגי, ומורפולוגיה, הקשורים הן לסימביונטים והן לאלמוגים המארחים. בנוסף, מחקר זה מאפשר התבוננות אל מערכת סימביוזה חדשה, בה שותפים אלמוגים , תולעים (Acoela) ואצות חד-תאיות.

החלק הראשון של המחקר עסק בשלב יצירת הקשר בין האלמוג הרך Heteroxenia fuscescens ובין האצות השיתופיות מהסוג Symbiodinium תוך ניסיון למצוא חלבונים המתבטאים כתלות במצב הסימביונטי אצל המארח. מערכת אידאלית למחקר של שלב יצירת הסימביוזה בין אלמוגים ואצות היא כזו, המאפשרת קבלה של דרגות צעירות חסרות ובעלות אצות שיתופיות. אלמוג עם העברה הוריזונטלית כדוגמת H. fusecescens הוא הכרחי למחקר כזה. בחלק זה של המחקר נעשה לשימוש בג' ים דו-מימדיים להפרדת חלבונים להשוואה בין פרופיל חלבונים של פוליפים ראשוניים, בני אותו גיל, שנבדלים במצבם הסימביונטי. זוהי העבודה הראשונה, שעוסקת בחיפוש חלבונים, המתבטאים בשלב יצירת הסימביוזה בין אלמוגים ואצות. הפרופילים שהתקבלו הראו שינויים בביטוי חלבונים כתלות בדרגה התפתחותית אך, למרבית ההפתעה, לא הראו שינויים בביטוי חלבונים כתלות במצב הסימביונטי. ייתכן, כי מתרחשים שינויים שלא התגלו במערכת שנבחנה כתוצאה מהסיבות שמפורטות להל"ן. ייתכן, כי כמות החלבונים קטנה ביותר וקצב ייצורם איטי ולכן הם אינם באים לביטוי בג'לים. ייתכן גם, כי ביטוי החלבונים מוגבל לתאים, שמכילים את הסימביונטים ומכיוון, שכמותם של האחרונים קטנה יחסית, הם "נבלעים" ברקע של חלבונים אחרים שמיוצרים בתאים הרבים האחרים. לסיכום, יצירת הסימביוזה בין האלמוג H. fuscescens והאצות השיתופיות שלו, ככל הנראה לא מלווה בשינויים גדולים בביטוי חלבונים בשבועות הראשונים שלאחר יצירת הסימביוזה.

חלקו השני של המחקר בדק את זהות האצות השיתופיות ברמה של Clade במגוון מיני אלמוגים רכים בהקשר לדרך העברת הסימביונטים שלהם, תוך שימוש בשיטת ה - RFLP של הגן לתת-היחידה הקטנה של ה - RNA הריבוסומלי. החלק השני, עסק בחיפוש אחר חלבונים, שקשורים לשלב יצירת הסימביוזה בין האלמוג הרך Heteroxenia fuscescens והאצות השיתופיות, תוך שימוש בג'לים דו-מימדיים להפרדת חלבונים. לבסוף, בחלקים השלישי והרביעי של המחקר, נבדקו היבטים שונים של מערכת הסימביוזה המשולשת הכוללת אלמוגים, תולעים ואצות שיתופיות. בנוסף נלמד תהליך הרבייה המינית ודרך העברת הסימביונטים בתולעת מהמין החדש Waminoa brickneri, שהוגדר במהלך עבודה זו. תוצאות החלק השני של המחקר הראו זו הפעם הראשונה, שקיים קשר בין דרך העברת האצות וזהות האצות במארח מסוים. נמצא, כי כל האלמוגים בעלי העברה הוריזונטלית של סימביונטים הכילו אצות השייכות ל - Clade C בעוד אלה בעלי העברה ורטיקלית הכילו סימביונטים השייכים ל - Clade A בלבד. קיימות עדויות ממחקרים שנערכו באיים הקאריביים, שאצות מ - Clade A בעלות התאמות יחודיות לחיים במים רדודים, עמידות בתנאי סביבה קיצוניים, בטווח טמפ' רחב והן היחידות המסוגלות לייצר חומרים המגינים מפני קרינת MAAs) UV). מחקרים אלה תומכים בהשערה, כי אצות מ - Clade A, המותאמות לחיים בתנאי סביבה משתנים, התפתחו במהלך האבולוציה כמותאמות ביותר להיות מועברות בהעברה

2 ורטיקלית, שכן הם הקנו למאחסנים שלהן עמידות בטווח רחב יחסית של תנאים ולכן שרדו לאורך זמן. הבלעדיות של Clade A במארחים עם העברה ורטיקלית באילת, היא ככל הנראה תוצאה של תהליך ויחסי קואבולוציה של המארחים והסימביונטים. הסימביונטים מ - Clade C, המאופיינים בהטרוגניות רבה יותר מבחינה גנוטיפית, נמצאו במארחים עם העברה הוריזונטלית וככל הנראה כל אחד מהגנוטיפים השונים הוא בעל סט תכונות פיזיולוגיות ייחודי ומצומצם יותר. בהסתמך על תוצאות מחקר זה ותוצאות מחקרים נוספים, שעסקו באלמוגי האבן Montipora ו - Porites שגם הם בעלי העברה ורטיקלית, אני משערת שאלמוגים בעלי דרך העברה זו, יינטו להכיל סימביונטים מגנוטיפים ייחודיים, השונים מאלה המצויים באלמוגים בעלי העברה הוריזונטלית. במחקר הנוכחי נמצא כי הסימביונטים נבדלו בשתי מערכות ההעברה בשייכותם ברמת ה - Clade. מושבות אלמוגים "מנוקדות" נצפו באילת כבר לפני יותר מעשור. רק לאחרונה יוחסה תופעה זו להימצאותן של תולעים ממחלקת ה - Acoela ומהסוג Waminoa. במהלך המחקר הנוכחי הוגדר המין האילתי כמין חדש למדע מסוג זה ונקרא Waminoa brickneri. ארבעה עשר מיני אלמוגים נמצאו כמאוכלסים ע"י התולעים, מתוכם 13 מיני אלמוגי אבן (גושיים ושיחניים) ואלמוג רך אחד. כל האלמוגים הכילו אצות שיתופיות. התולעים הכילו שני טיפוסי אצות שיתופיות: טיפוס קטן שזוהה כשייך לסוג Symbiodinium וטיפוס גדול יותר, שככל הנראה שייך לסוג Amphidinium. בהשוואה שנערכה באמצעות מיקרוסקופ אלקטרונים סורק, בין ענפי מושבה שהיו מכוסים בתולעים לבין כאלה שהיו נקיים, נמצא כי באחרונים שכבת ריר כיסתה את כל פני השטח לעומת זאת בראשונים פני השטח היו נקיים לחלוטין מריר. באנליזה מולקולרית השוואתית (DGGE) של האצות השיתופיות מהסוג Symbiodinium, שבודדו מהאלמוגים ומהתולעים שנמצאו עליהם, נמצא כי ברוב המוחלט של המקרים, הגנוטיפ של האצות מהאלמוגים היה שונה מזה של האצות מהתולעים. על כן, סביר להניח, כי התולעים לא משיגות את האצות השיתופיות על ידי טריפה של רקמת האלמוג. המעקב אחר הרבייה המינית של תולעים מהמין Waminoa brickneri באמצעות חתכים היסטולוגיים, מיקרוסקופיה אלקטרונית חודרת ומעקב במעבדה הראה כי באואוציטים של תולעים בוגרות מבחינה מינית קיימת נוכחות של תאי אצה משני הטיפוסים. ממצא זה מצביע על כך, כי התולעים מעבירות את האצות השיתופיות בהעברה ורטיקלית. זהו התיעוד הראשון של דרך העברת אצות כזו באורגניזם תלת-שכבתי. ממצא זה מלמד, כי התולעים מעבירות את הסימביונטים מדור לדור מבלי להיות תלויות במקור חיצוני כלשהו, כדוגמת האלמוג המארח, כמקור לסימביונטים. בנוסף התגלה כי ההתפתחות במין זה היא ישירה ומהביצה בוקעת תולעת זעירה ושחיינית. תוצאות מחקר זה מעודדות המשך מחקר אינטנסיבי ומקיף, שיעסוק באלמוגים שמעבירים את הסימביונטים שלהם בהעברה ורטיקלית וזאת כדי להבין את המנגנונים, המאפשרים את קיומם של אלמוגים אלה בסביבת חיים משתנה. יתרה מכך, יהיה מעניין ללמוד על היכולות הפיסיולוגיות של הסימביונטים המועברים בהעברה כזו. התוצאות השליליות שהתקבלו בפרק שעסק בחלבונים תלויי-סימביוזה מרמזות על הצורך בשימוש בשיטות שונות בעלות רזולוציה טובה יותר במחקר עתידי שיעסוק בנושא זה. לבסוף, חשיפת מערכת הסימביוזה המשולשת המערבת אלמוגים, תולעים ואצות חד תאיות, כפי שהתגלתה בשונית

3 של אילת, ללא ספק תשמש כבסיס למחקרים נוספים, שיבחנו היבטים של ספציפיות, פיסיולוגיה ומגוון השותפים במערכת מיוחדת זו.

4

מערכות סימביוזה ימיות הכוללות אלמוגים , תולעים ואצות

חד-תאיות: ראשית הסימביוזה, ספציפיות,

מגוון הסימביונטים ואופן רכישתם

חיבור לשם קבלת התואר "דוקטור לפילוסופיה"

מאת

אורית ברנע

הוגש לסנאט אוניברסיטת תל-אביב

אב תשס"ה

אוגוסט 2005

מערכות סימביוזה ימיות הכוללות אלמוגים, תולעים ואצות

חד-תאיות: ראשית הסימביוזה, ספציפיות,

מגוון הסימביונטים ואופן רכישתם

חיבור לשם קבלת התואר "דוקטור לפילוסופיה"

מאת

אורית ברנע

הוגש לסנאט אוניברסיטת תל-אביב

אב תשס"ה

אוגוסט 2005

עבודה זו נעשתה בהדרכת

פרופ' יהודה בניהו

ופרופ' וירג'יניה וייס