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BIOACTIVE MOLLUSCAN RESOURCES

AND THEIR CONSERVATION:

Biological and Chemical Studies on the Egg Masses of

Marine Molluscs

* A thesis submitted in fulfilment of the

requirement for the award of the degree

DOCTOR OF PHILOSOPHY

from

UNIVERSITY OF WOLLONGONG

KIRSTEN BENKENDORFF, B.Sc. Hons.

Department of Biological Sciences Department of Chemistry

March, 1999

The edge of the sea is a strange and beautiful place.

Rachael Carson

Certainly Divinity is here in these shells in their humble form of life.

Frank Lloyd Wright

By Tyre the old, with ocean-plunder, A netful, brought to land.

Robert Browning

Dedicated to my Grandmother, Jutta Benkendorff

For inspiring me to take a philosophical approach to evolution,

And my parents, Robin and Peter Benkendorff

For endless support and encouragement.

III

Thesis Declaration

This thesis contains no material that has been accepted for the award of any other degree or diploma at any University, and to the best of my knowledge contains no

material previously published or written by another person, except where due

reference is made in the text of the thesis.

Acknowledgments

I am indebted to Dr Andy Davis and Prof. John Bremner from the University of

Wollongong for supervising this project. I would like to especially thank Dr Andy Davis

(Department of Biological Sciences) for initially suggesting that I study the egg masses of marine molluscs. Having never before studied in the marine environment, Andy’s knowledge of marine biology has greatly assisted me during this project. Andy has also significantly improved my understanding of experimental design, as well as providing enthusiasm, encouragement and valuable comments throughout the research project. I am also extremely grateful to Prof. John Bremner (Department of

Chemistry) for agreeing to take on a biology student and making available his knowledge of organic and natural products chemistry. John’s advice has been essential to some aspects of this research project and his comments on all aspects of the study have been valuable and are much appreciated.

Sincere thanks are extended to my parents for, in the words of my mother, “providing a good set of genes” but perhaps more importantly, for providing the perfect environment in which to grow and learn. I would also like to thank the rest of my family, particularly Carlo Pisanu, for being supportive and accepting my asocial behaviour over the last few years.

I am grateful to Dr. Bill Rudman, Mr Ian Loch and Dr. Winston Ponder from the

Australian Museum, for assisting me with species identification and for generally improving my knowledge of marine molluscs. I have also appreciated the

V opportunity to access the wonderful resources (specimens and literature) that are available in the Malacology Department of the Australian Museum.

I am grateful to Dr. John Korth from the University of Wollongong (UOW, Chemistry

Department) for assisting me on the GC/MS. Thanks also to Dr. Renate Griffiths from the Department of Chemistry (UOW) for performing the conformational search on

Tyriverdin in the SPARTAN modelling program. Darren Saunders (Department of

Biological Sciences, UOW) kindly cultured the human lymphoma cells that were use in this project and explained the methods used for cytotoxicity testing. My appreciation is extended to Mr. George Gray and Dr. Alistair Lochhead from Southern Pathology,

Wollongong, NSW, for the use of the cytospin and light microscope/camera equipment. Dr. David Muir from the Royal North Shore Hospital kindly provided two strains of Candida albicans and Dr Jeremy Carson from the Fish Health Unit,

Department of Primary Industry and Fisheries, Tasmania, provided the strains of marine pathogens.

I would like to further thank Dr Andy Davis, for providing the egg masses of a subtidal nudibranch, a terrestrial snail and several Mediterranean molluscs. The assistance of

Dr Manual Ballesteros (University of Barcelona, Spain) in the collection of the egg masses from Spurilla neopolitana and Ceratostoma erineceum is also appreciated.

The Australian specimens collected for this study were authorised under a general scientific permit for the collection of marine invertebrates outside

VI reserve areas in the waters of N.S.W (reference no. F95/269). This license was authorised in accordance with the provisions of Section 37 of the Fisheries

Management Act 1994.

The provision of an Australian Postgraduate Award from the Australian Government, as well as a Supplementary Postgraduate Scholarship from the Australian Flora and

Fauna Research Centre (AFFRC), the Bioactive Molecules Research Centre and the

Environmental Research Institute at the University of Wollongong is greatly appreciated. I would also like to thank the members of the AFFRC and John

Bremner’s research group for interesting discussions.

My most sincere thanks to all

Kirsten Benkendorff, 1999

VII

Abstract

Chemical prospecting for pharmaceuticals in natural organisms (bioprospecting) can be used as a tool for the conservation of biological diversity. However, bioprospecting can only be considered compatible with conservation if it is conducted in an environmentally sustainable manner. In order to prevent the overcollection of vulnerable organisms it is essential to gain an understanding of the local distribution and abundance of the target organisms. In this study, the egg masses of intertidal molluscs were targeted as a novel source of biologically active compounds. Surveys of the molluscan fauna were conducted on 13 intertidal reefs along the Wollongong

Coast, New South Wales, Australia. In total, 161 species of intertidal molluscs were found and the benthic egg masses from 47 species were identified. Only 31% of these molluscs have been previously recorded from intertidal surveys in the region and 66% of the species may be regarded as regionally rare. Repeated surveys of the 13 reefs revealed that the species diversity recorded in a single inventory was representative of the cumulative diversity detected. ‘Hotspots’ of molluscan diversity were found on the northern side of two large headlands (Bass Point and Bellambi Point), which are characterised by a high habitat complexity and shelter from strong wave action.

Three selective pressures could potentially lead to the evolution of chemical defence in molluscan egg masses: predation, disease and surface fouling. Marine molluscs may rely on a range of alternative strategies to protect their egg masses from predators, including physical protection in leathery egg capsules, camouflage and rapid embryonic development, as well as behavioural mechanisms, such as brooding and the deposition of large aggregated egg masses. Predator feeding trials provided evidence of chemical defence in five out of eight species that were tested. On the other hand, observational studies provided no evidence to suggest that molluscan egg

VIII masses are chemically defended against surface fouling by macroorganisms. A range of macrophytes and epizooites were observed on the surface of both gelatinous egg masses and leathery egg capsules. Nevertheless, the overall incidence of fouling was low, probably because of their ephemeral nature and the fact that most molluscs deposit egg masses on the underside of boulders.

Disease appears to be a significant selective pressure leading to the evolution of chemical defence in molluscan egg masses. Two assays were used to screen the egg masses of marine molluscs for antimicrobial activity against human and marine pathogens; a modified version of the traditional Zone of Inhibition assay and the

Fluorescein Diacetate assay. These two assays have small sample requirements and thus it was possible to screen the egg masses of 42 molluscs and four polychaetes.

Antimicrobial activity against at least one human pathogen was found in the egg masses of 36 species, including two polychaetes and a wide range of molluscs. The egg masses from a number of species clearly lose activity during embryonic development. The antimicrobial activity also appears to be greater in the internal matrix, rather than on the outer surfaces of molluscan egg masses. Surface bacteria could be responsible for the observed activity in some species but are unlikely to be the source of antimicrobial agents in leathery egg capsules, or the gelatinous egg ribbons of Aplysia spp. The egg masses of Dicathais orbita and Aplysia juliana were found to inhibit ecologically significant marine bacteria, as well as Gram negative and

Gram positive human pathogenic bacteria and the yeast Candida albicans.

The compounds responsible for the antimicrobial activity in the egg masses of the common muricid Dicathais orbita were isolated using bioassay-guided fractionation.

These were then identified by mass spectrometry and proton nuclear magnetic

IX resonance spectroscopy. Three antimicrobial compounds were characterised

(tyrindoleninone, tyriverdin and 6-bromoisatin) and these are all known precursors to the ancient dye Tyrian Purple. Tyrindoleninone is the most abundant volatile organic compound found in the fresh eggs and this compound was shown to be toxic to bacteria at a concentration of 1mg/ml. As the eggs develop, most of the tyrindoleninone is converted into tyriverdin, which was found to be effectively bacteriostatic at 0. 5 µg/ml but was not cytotoxic at 1 mg/ml. This compound is considered to be a useful new drug lead. The 6-bromoisatin, which is likely to be an oxidative artefact derived from the other precursors, exhibited mild cytolytic activity against a range of bacteria. As the larvae began hatching, most of the tyriverdin was converted into Tyrian Purple in the egg masses. Tyrian Purple did not exhibit any significant antimicrobial activity, although it is highly insoluble in aqueous media.

Nevertheless, these studies provide evidence for a chemical ripening process in the egg masses of Dicathais orbita, which may provide a means of avoiding autotoxicity to the larvae during hatching.

Extracts from the egg masses of 23 molluscs were then examined for the precursors of Tyrian Purple, as well as other potential antimicrobial agents, using gas chromatography/ mass spectrometry. The egg masses from six species of Muricidae were found to contain the precursors of Tyrian Purple. However, these compounds were not found in the egg masses of species from any other family. A range of other related indoles, as well as di- and tribromoimidazoles/pyrazoles were also found in the egg masses of the Muricidae. Most of these compounds have not been previously described from a natural source and they could all contribute to the observed antimicrobial activity in the muricid egg masses. The egg masses of the Aplysiidae were found to contain some bioactive polychlorinated hydrocarbons and a range of

X long chain unsaturated fatty acids. Halogenated compounds were not found in the egg masses of any other species, although fatty acids could be partly responsible for the observed antimicrobial activity in most of the gelatinous egg masses. A high diversity of volatile organic compounds was found in the molluscan egg masses, but further work is required to identify the active components.

Clearly, bioprospecting can contribute to conservation through the development of comprehensive species inventories. Bioprospecting can be conducted with minimal impact on the environment and the discovery of novel bioactive compounds provides an incentive for conservation. All marine molluscs that deposit benthic egg masses have potential pharmaceutical value and therefore an effort should be made to conserve both them and their natural habitats. Bass Point would be an appropriate site for an intertidal protected area in the Wollongong region.

Table of Contents

Acknowledgments V Abstract VIII

CHAPTER 1 1 MOLLUSCAN RESOURCES AND CONSERVATION 1.1 General introduction 1 1.2 Molluscan resources 2 1.2.1 The diversity of resources. 2 1.2.2 Bioactive molluscan resources. 3 1.3 The biorational approach to drug discovery 7 1.3.1 The biorational approach. 7 1.3.2 Egg masses as targets for drug discovery. 9 1.3.3 Molluscan reproductive strategies. 10 1.4 Bioprospecting 14 1.4.1 Conservation and bioprospecting. 14 1.4.2 The ethics of bioprospecting. 15 1.4.3 Is bioprospecting environmentally sustainable? 16 1.4.4 Requirements for sustainable bioprospecting. 17 1.5 Molluscan conservation 20 1.5.1 The conservation status of marine molluscs. 20 1.5.2 Habitat protection. 21 1.6 Structure of this study 22

CHAPTER 2 25 MOLLUSCAN DIVERSITY: SPECIES INVENTORIES AND HABITAT ASSESSMENT 2.1 Introduction 25 2.1.1 Species diversity, species rarity and conservation implications. 25

XII

2.1.2 Intertidal reefs and the determinants of species diversity. 28 2.1.3 Species inventories and rapid biodiversity assessment. 31 2.1.4 Molluscs of the Illawarra Coast. 33 2.2 Objectives 38 2.3 Methods 38 2.3.1 Study sites. 38 2.3.2 Surveys. 43 2.3.3 Habitat quality. 47 2.3.4 Statistical analysis. 50 2.4 Results 52 2.4.1 Species list of intertidal molluscs in the Wollongong region. 52 2.4.2 Comparisons to previous surveys. 59 2.4.3 Natural reefs vs. artificial reefs. 61 2.4.4 Egg laying habitats. 61 2.4.5 Spatial and temporal variation in molluscan diversity. 64 2.4.6 Molluscan diversity and the physical environment. 68 2.5 Discussion 72 2.5.1 Molluscan diversity and distribution in the Wollongong region. 72 2.5.2 Species rarity and implications for resource management . 73 2.5.3 Molluscan breeding habitats. 78 2.5.4 Molluscan diversity and the physical environment. 80 2.5.5 Methods for assessing intertidal molluscan diversity. 82 2.5.6 Intertidal management recommendations. 89 2.6 Conclusion 90

CHAPTER 3 93 DEFENCE AGAINST PREDATION AND MACRO FOULING ON MOLLUSCAN EGG MASSES 3.1 Introduction 93

XIII

3.1.1 Marine predators and predation on molluscan egg masses. 93 3.1.2 Adaptations to predation: Behavioural and physiological defence. 95 3.1.3 Physical defence against predation. 96 3.1.4 Chemical defence mechanisms. 97 3.1.5 Predator feeding trials for assessing the role of chemical defence. 98 3.1.6 Macro fouling. 101 3.2 Objectives 104 3.3 Methods 104 3.3.1 Field and laboratory observations. 104 3.3.2 Collection and maintenance of predators. 105 3.3.3 Collection and preparation of egg material. 107 3.3.4 Preparation of artificial feeding disks. 107 3.3.5 Crab feeding experiments. 109 3.3.6 Isopod feeding experiments. 110 3.3.7 Starfish feeding trials 111 3.3.8 Field feeding trials 112 3.3.9 Statistical analysis 113 3.4 Results 115 3.4.1 Defensive strategies against predation. 115 3.4.2 Chemical defence: Crab feeding trials. 119 3.4.3 Chemical defence: Isopod feeding trials. 120 3.4.4 Chemical defence: Field feeding trials. 124 3.4.5 Starfish feeding trial. 125 3.4.6 Physical defence. 127 3.4.7 Macrofouling. 128 3.5 Discussion 131 3.5.1 Defensive strategies against predation. 131 3.5.2 Chemical defence and artificial feeding trials. 134 3.5.3 Physical defence against predation. 139 3.5.4 Macrofouling on molluscan egg masses. 140 3.6 Conclusion

XIV

CHAPTER 4 143 ANTIMICROBIAL ACTIVITY IN MOLLUSCAN EGG MASSES 4.1 Introduction 143 4.1.1 The need for novel antibiotics. 143 4.1.2 Antimicrobial activity in molluscs and molluscan egg masses. 144 4.1.3 Assays for detecting antimicrobial activity. 147 4.1.4 Microfouling and symbiosis. 155 4.2 Objectives 157 4.3 Methods 158 4.3.1 Collection and preparation of egg material. 158 4.3.2 Maintenance and preparation of microbial cultures. 162 4.3.3 Zone of Inhibition assay. 164 4.3.4 The Fluorescein Diacetate (FDA) assay. 165 4.3.5 Antimicrobial (cell lysis/ cell stasis) assay. 167 4.3.6 Microfouling and symbiosis. 168 4.4 Results 168 4.4.1 Antimicrobial activity in benthic invertebrate egg masses. 168 4.4.2 Antimicrobial properties at different stages of development. 169 4.4.3 Zone of Inhibition assay. 172 4.4.4 Fluorescein Diacetate assay. 178 4.4.6 Antimicrobial (cell stasis/lysis) activity. 186 4.4.7 Epibiosis and microfouling. 191 4.5 Discussion 192 4.5.1 Antibiotics in invertebrate egg masses. 192 4.5.2 The resource potential of molluscan antibiotics. 195 4.5.3 Developmental changes in antimicrobial properties. 197 4.5.4 Localisation of the antimicrobial components and autotoxicity. 199 4.5.5 Properties of the antimicrobial components. 202 4.5.6 Screening methods for detecting antimicrobial activity. 204 4.5.7 Epibiosis and symbiosis. 208

4.6 Conclusion 210

CHAPTER 5 213 ISOLATION AND CHARACTERISATION OF THE ANTIMICROBIAL COMPOUNDS FROM THE EGG MASS OF DICATHAIS ORBITA 5.1 Introduction 213 5.2 Objectives 219 5.3 Methods 219 5.3.1 Collection and extraction of egg masses. 219 5.3.2 Analysis of the crude extract. 220 5.3.3 Isolation and identification of antimicrobial components. 221 5.3.4 Antimicrobial testing. 222 5.3.5 Cytotoxicity testing. 223 5.4 Results 224 5.4.1 Analysis of the crude egg extracts. 224 5.4.2 Antimicrobial activity. 229 5.4.3 Cytotoxicity of tyriverdin. 231 5.5 Discussion 238 5.5.1 Antimicrobial compounds from the egg mass of Dicathais orbita. 238 5.5.2 Tyriverdin – a novel drug lead. 241 5.6 Conclusion 248

CHAPTER 6 251 VOLATILE ORGANIC COMPOUNDS IN MOLLUSCAN EGG MASSES 6.1 Introduction 251 6.1.1 Indole derivatives from muricids. 253 6.1.2 Other bioactive halogenated compounds from molluscs. 255 6.1.3 Nonhalogenated bioactive compounds from molluscs. 259 6.1.4 Metabolites from molluscan egg masses. 260 6.1.5 Chemical diversity and dereplication. 264 6.2 Objectives

XVI

6.3 Methods 266 6.3.1 Specimen collection and sample preparation. 266 6.3.2 Derivatisation procedure. 267 6.3.3 Gas chromatography/mass spectrometry analyses. 267 6.3.4 Antimicrobial activity of identified egg constituents. 268 6.4 Results 270 6.4.1 Brominated compounds in muricid egg masses. 270 6.4.2 Other related compounds from muricid egg masses. 277 6.4.3 Halogenated compounds from Aplysiidae egg masses. 279 6.4.4 Fatty acids and methyl esters. 284 6.4.5 Other volatile components of molluscan egg masses. 286 6.4.6 Antimicrobial activity of some common egg metabolites. 294 6.5 Discussion 296 6.5.1 Bioactive compounds from muricid egg masses. 296 6.5.2 Bioactive metabolites in Aplysiidae egg masses. 301 6.5.3 Bioactive metabolites from other molluscan egg masses. 304 6.6 Conclusion 307

CHAPTER 7 GENERAL DISCUSSION 309 BIOACTIVE MOLLUSCAN RESOURCES AND CONSERVATION 7.1 Biodiversity and bioresources 309 7.2 The biorational approach to drug discovery 312 7.3 An interdisciplanary approach 314 7.4 Sustainable bioprospecting 317 7.5 Bioprospecting and conservation 319 7.6 Management recommendations 321 7.7 Conclusion 323

REFERENCES 325 CHAPTER 1: APPENDIX 1 383

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Appendix 1.1 383 Appendix 1.2 390 CHAPTER 2: APPENDIX 2 397 Appendix 2.1 398 Appendix 2.2 431 Appendix 2.3 441 CHAPTER 4: Appendix 4 445 Appendix 4.1 445 Appendix 4.2 446 Appendix 4.3 448 CHAPTER 6: Appendix 6 451 Appendix 6.1 452 Appendix 6.2 515 Appendix 6.3 526 Appendix 6.4 527 Appendix 6.5 531 Appendix 6.6 534 Appendix 6.7 551 Appendix 6.8 553

XVIII

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UNIVERSITY OF WOLLONGONG

Chapter 1

Introduction: Molluscan Resources and Conservation

“All we have yet discovered is but a trifle in comparison to what lies hid in

the great treasury of nature”

Antoni van Leewenhoek, 1680

1.1 General introduction

Continued pressure on the environment from technological progress and human population growth has made it necessary to place a commodity value on biodiversity (Randall, 1991). The provision of unprecedented biologically active compounds is one value of biodiversity that has been widely recognised

(Eisner, 1990; Wilson, 1994; Beattie, 1994). As stated by Thomas Eisner (1990)

“In a very real sense, and quite aside from other measures of worth, species have chemical value.” The large returns from the sale of naturally derived pharmaceuticals has provided an incentive for some companies to channel money back towards the conservation of biodiversity (Roberts 1992; Carté

1996). Thus, within the pharmaceutical industry there has been some recognition of the fact that the continued discovery of new chemical products depends on the maintenance of biological diversity.

Over the last decade there has been a resurgent interest in the investigation of natural products as a source of new pharmaceutical agents (Beattie, 1994;

Cragg et al., 1997). This has been sparked by growing problems such as, the evolution of resistance to antibiotics, the increased occurrence of some Chapter 1: Introduction: Molluscan resources and conservation 2

diseases (e.g. cancer) and the evolution of new diseases (e.g. HIV). Coupled with this is the rapid rate of species extinction and the continuing threat to biodiversity. Recognition of these factors lead to the development of the

Göteborg Resolution by the International Society of Chemical Ecology, which calls for an increased effort to expand the search for new natural products

(Eisner and Meinwald, 1990).

This study involves searching for a new source of antibiotics in the egg masses of marine molluscs. The purpose of this Chapter is to provide some background information on the resource potential and conservation status of marine molluscs. The biorational approach to drug discovery will then be used to explain the rationale behind targeting molluscan egg masses as a source of new biologically active compounds. The conservation incentives and environmental problems surrounding chemical prospecting in natural organisms will also be discussed.

1.2 Molluscan Resources

1.2.1 The diversity of resources Throughout history, in all parts of the world, marine molluscs have provided a diverse range of resources, including food, pearls, shell grit, ornaments, jewellery, utensils and musical instruments. Shells have been used as a commercial currency, traded and offered as gifts to emperors and Gods (Stix et al. 1978; Wye 1996). Marine molluscs in the family Murcidae were particularly treasured in ancient Roman, for the production of a deep purple dye known as

Tyrian Purple (Baker, 1974). The aesthetic qualities of shells have also inspired Chapter 1. Introduction: Molluscan resources and conservation 3 artists and architects (Stix et al., 1978) and more recently the opisthobranchs, shell-less molluscs, have provided a favourite subject for underwater photography.

Molluscs are the second largest animal phylum on earth and include an enormous diversity of species (Beesley et al., 1998). However, the resource potential of most molluscs is likely to be underestimated. Human needs and values change, and consequently, the value of natural resources can also change over time (Benkendorff, 1999). Our general perception of what constitutes a resource may be too narrow. All species are a potential source of information and could therefore enable the discovery and development of new resources. In particular, the vast diversity of molluscs in the marine environment provides an enormous resource for natural products research.

1.2.2 Bioactive molluscan resources A number of reviews have been specifically dedicated to natural products isolated from marine molluscs (Faulkner, 1984a; Karuso, 1987; Faulkner,

1992a; Myers et al., 1993; Avila, 1995; Roseghini et al., 1996). An annual review of the literature on marine natural products by Faulkner (1984a,b,

1986, 1987, 1988, 1990, 1991, 1992b, 1993, 1994, 1995b, 1996, 1997) cites a total of 369 papers on molluscan natural products. Significantly, Faulkner’s reviews do not provide a comprehensive coverage of all the literature on marine natural products, instead they focus on marine natural products with interesting biological and pharmaceutical properties.

Chapter 1. Introduction: Molluscan resources and conservation 4

By far the majority of research on natural products from the Mollusca has been in the class Gastropoda. One group of gastropods in particular, the

Opisthobranchs, have attracted much attention from natural products chemists. Over 70% of the studies on marine molluscs cited in Faulkners’ reviews, have concentrated on the Opisthobranchia. Nevertheless, Avila

(1995) suggests that the chemistry of only 249 of the approximately 6000 described species of Opisthobranchs has been studied.

The relatively large representation of the order Opisthobranchia in marine natural products research can be explained by the fact that these are primarily soft-bodied or shell-less molluscs. It is generally assumed that the presence of a shell provides protection from predation and therefore shelled molluscs do not possess any special chemicals that could function in defence (refer to

Faulkner, 1992a). Notably, most Cephalopods also lack an external shell, although few studies have reported natural products from this class of molluscs. It has been suggested that the great speed and the release of ink compensates for the apparent physical vulnerability of Cephalopods

(Faulkner, 1992a). However, the dearth of literature on molluscs other than the Opisthobranchs is most likely due to the bias towards collecting these species, rather than the absence of biologically active substances in other molluscan taxa.

The natural products isolated from marine molluscs have been tested for a broad range of biological activities. Molluscan metabolites have been most commonly tested for neuromuscular blocking action, antipredator,

Chapter 1. Introduction: Molluscan resources and conservation 5 antimicrobial, antineoplastic and cytotoxic activity (Figure 1.1). A summary of

51 references from the literature on molluscan metabolites, found only seven cases where a species did not show activity in a particular assay (Figure 1.1;

Appendix 1.1). This suggests that molluscan metabolites have a high incidence of biological activity. However, two shipboard screening expeditions by Rinehart et al., (1981) found that only a small proportion of molluscs exhibited pharmacological activity (Figure 1.2). Consequently, the high incidence of biological activity reported in the general scientific literature on molluscs is probably due to the fact that negative results are unfortunately, rarely published. Alternatively, it could be due to the fact many studies target species that are most likely to be chemically rich.

Perhaps the most promising metabolite isolated from a marine mollusc is

Dolastatin 10, an antineoplastic peptide isolated from the sea hare Dolabella auricularia (Pettit et al., 1987). The medical properties of this sea hare were known to the ancient Greeks and Romans, with the extracts being recommended for the treatment of some diseases as early as 200BC (refer to

Pettit et al., 1987). Dolastatin 10 has recently reached clinical trials in the

United States and is reported to be one of the most potent anticancer agents known (Carté, 1996). Toxic peptides produced by predatory snails in the genus Conus also provide a valuable resource. These toxins are being used as tools for neurological research (Carté 1996) and are being trialed as strong pain killers and potential drugs against epilepsy, depression and schizophrenia (Concar 1996). These two examples demonstrate the potential for discovering commercially viable metabolites from marine molluscs.

Chapter 1. Introduction: Molluscan resources and conservation 6

60 Active Not active

30 Number of species

0 Antimicrobial/ Neuromuscular Cytotoxic/static Antineoplastic Ichyotoxic/ Toxic to mice Other antifungal blocking antifeedant

Figure 1.1: The biological activity of metabolites from 120 molluscs reported in 51 references listed in Appendix 1.1.

200

180 Active Not active 160

140

120

100

80 Number of species

0 Antimicrobial Cytotoxic Antiviral Figure 1.2: The number of molluscs showing biological activity in two shipboard screening expeditions (Rinehart et al., 1981).

Chapter 1. Introduction: Molluscan resources and conservation 7

1.3 The biorational approach to drug discovery

“Millions of years of testing by natural selection have made

organisms chemists of superhuman skill, champions of

defeating most of the kinds of biological problems that

undermine human health”

E.O. Wilson, 1994

1.3.1 The biorational approach The International Society of Chemical Ecology has indicated the need for increased “biorational” studies aimed at discovering new biologically active chemicals (Eisner and Meinwald, 1990). It has been estimated that 98 per cent of marine samples collected by bioprospectors are discarded before there has been any detailed chemical or pharmacological analysis (Anderson,

1995; Garson, 1996). The biorational approach to the discovery of new natural products has been described by Beattie (1994) and involves the use of biological data to target those species most likely to yield an appropriate natural product. Ultimately, this approach should narrow the field of search to manageable proportions and increase the likelihood of discovering a useful natural product.

In the marine environment much of the natural products research has concentrated on sessile invertebrate phyla, which have no means of physical defence (Scheuer, 1990; de Vries and Hall, 1994; Flam, 1994; Faulkner,

1995a; Liles, 1996). Notably, the richest source of drugs from the terrestrial

Chapter 1. Introduction: Molluscan resources and conservation 8 environment are derived from plants (Cragg et al., 1997), which are also sessile and have therefore evolved chemical defence mechanisms against predators and parasites. Sessile marine organisms have evolved toxic compounds to avoid predators, as well as to compete for space and keep their surfaces free from fouling organisms (reviewed by Bakus et al., 1986;

Paul, 1990; Pawlik, 1993). This biorational approach to marine natural products chemistry was adopted in the 1970s by some of the early leaders in the field, for example the Roche Research Institute of Marine Pharmacology

(Baker, 1976) and the Scripps Institution of Oceanography (Scheuer, 1989).

The biorational approach has been loosely applied to the discovery of a range of bioactive compounds in the marine environment. Antimicrobial, cytotoxic, antitumor, tumour-promoting, anti-inflamatory and antiviral agents have all been isolated from sessile, soft-bodied marine organisms (Sheuer, 1989;

Schmitz and Yasumoto, 1991; Carté, 1996). The chemicals that have evolved to protect these benthic organisms are intrinsically bioactive and are often highly active in too many pharmacological assays to be used directly as drugs

(Faulkner, 1995a). Clearly, a more focussed use of the biorational approach in the marine environment has limitations for many diseases that are specific to mammals. However, the biorational approach is particularly suited to searching for novel antimicrobial agents in the marine environment, because benthic organisms are living in a soup of microorganisms (Austin, 1988).

In the search for a novel source of antibiotics it is appropriate to consider the circumstances under which antibiotics might be expected to evolve. Most

Chapter 1. Introduction: Molluscan resources and conservation 9 species of invertebrates suffer disease from a wide range of microbial pathogens and parasites (Austin, 1988). Certain levels of immunity can be found in all living things. However, invertebrates lack many features of the vertebrate immune system (Beck and Habicht, 1996) and studies on invertebrate immunity have revealed some novel types of defence, such as antibacterial peptides and proteins (e.g. Hoffmann and Hetru, 1992). On exposure to foreign material, some molluscs have been shown to exhibit a humoral defence response, including the release of bacteriostatic and bactericidal substances (reviewed by Tripp, 1975; Sminia and van der Knaap,

1985). Some of these molecules could have significant applications in medicine.

Contagious disease presents a particularly important problem to organisms that are obliged to live high densities (Beattie, 1994). In support of this rationale, a range of social insects have been shown to produce substances with antibiotic properties (Blum et al., 1959; Wilson, 1971; Maschiwitz, 1974;

Beattie et al., 1986; Veal et al., 1992; Mackintosh et al., 1994; Benkendorff,

1994). Many different kinds of animals aggregate during breeding season and these too may have adaptations to contagious disease (Beattie, 1994).

Species that deposit eggs in communal situations may also have evolved chemical protection specifically for their developing embryos.

1.3.2 Egg masses as targets for drug discovery As stated by Marsh and Rothschild (1974), “Eggs are sitting targets and it is in these organisms one should seek for toxic compounds”. Higher chemical

Chapter 1. Introduction: Molluscan resources and conservation 10 defence of reproductive tissue has been documented for a broad spectrum of species including plants (Thompson, 1982), bryozoa (Harvell, 1984) and terrestrial invertebrates (Blum et al., 1959; Snyder and Synder, 1971; Hare and Eisner, 1993). Coll et al., (1985) found that Australian soft corals

(Anthoza Octocorallia) synthesise two oxygenated diterpenes over the last few weeks prior to spawning and these compounds were only found in the eggs. At least one of these compounds appears to be toxic to fish, suggesting they may serve a role in chemical protection of the eggs (Coll, 1992).

Chemical defence has also been reported in the eggs of some echinoderms

(Hart et al., 1979; McClintock and Vernon, 1990), crustaceans (Gil-Turnes et al., 1989; Gil-Turnes and Fenical, 1992), grasshoppers (Euw et al., 1967) and jelly fish (Szollosi, 1969), as well as several vertebrate egg masses (reviewed by Orians and Janzen, 1974).

1.3.3 Molluscan reproductive strategies Many marine molluscs have evolved a reproductive strategy that involves depositing fertilised embryos in benthic egg masses. These egg masses appear to be subject to similar selective pressures as many of the sessile marine invertebrates that have evolved a chemical defence mechanism.

Molluscan egg masses can provide a considerable source of nutrients

(Pechenik, 1979; Gallardo, 1979; Stöckmann-Bosbach and Althoff, 1989) and developing eggs are generally thought to represent an attractive source of food (Orians and Janzen, 1974). Many molluscan egg masses are highly conspicuous (personal observation) and the development of some encapsulated embryos can last for many months (Laxton, 1969, West, 1973,

Chapter 1. Introduction: Molluscan resources and conservation 11

Havenhand, 1993). The colonisation of free surfaces in the marine environment occurs rapidly and every surface is a potential substrate for another marine organism (Davis et al., 1989). Consequently, predation and surface fouling could be significant selective pressures acting on the evolution of benthic egg masses.

Disease is also likely to be a significant selective pressure on molluscan egg masses. It is not known when the immune system becomes sufficiently well established to protect the embryos during molluscan development. However, it seems unlikely that newly fertilised eggs designate some of their limited nutritive reserves to the production of defensive compounds (refer to Orians and Janzen, 1974). Nevertheless, it is possible the adult molluscs enclose their fertilised eggs with antimicrobial compounds. Significantly, the egg masses of some marine molluscs show strong similarities to the brood cells of terrestrial invertebrates, such as honey bees, which have been shown to incorporate antimicrobial properties (Blum et al., 1959). All of the eggs within the spawn from one individual are genetically similar and close genetic relatedness would greatly exacerbate the impact of disease within the brood

(refer to Beattie, 1994). Behaviours such as communal spawning and the production of large aggregated egg masses would also facilitate the spread of an infectious disease. These breeding strategies have been reported for some marine molluscs (D’Asaro, 1991; 1993).

The egg masses of marine molluscs are generally thought to play a protective role (e.g. Thorson, 1950; Mileikovsky, 1971; Smith et al., 1989). The energy

Chapter 1. Introduction: Molluscan resources and conservation 12 expenditure associated with encapsulation appears to be high, suggesting that encapsulation must have substantial survival value (Pechenik, 1979).

Furthermore, the instantaneous mortality rates of benthic egg capsules appear to be much lower than estimated mortality rates for planktonic development (Strathman, 1985). Thorson (1950) suggests that the benthic egg masses of marine invertebrates provide protection against infection, although there is currently no data to support this. Pechenik (1979) suggests that the encapsulation may reduce developmental mortality by retaining developmental stages until they are better able to avoid predation.

Nevertheless, it is currently unclear how molluscan egg masses provide protection for developing embryos in the benthos.

Disease, predation and surface fouling could all reasonably lead to the evolution of chemical defence mechanisms. However, physical and behavioural mechanisms could also influence the variability in egg mass resistance to some sources of mortality. For example, parental site selection for the deposition of egg masses may be an important defence against predation and fouling, although these behavioural strategies are less likely to provide protection against disease. There are two major forms of molluscan egg masses, gelatinous egg masses and leathery egg capsules (Figure 1.3;

Smith et al., 1989). Leathery egg capsules appear to provide physical protection to the developing embryos against some marine predators (e.g.

Rawlings, 1990; 1994). The contents of leathery egg capsules also appear to be free of bacteria (Lord, 1986), although, it is presently unclear whether the capsule walls provide a physical barrier against microbial infection. On the

Chapter 1. Introduction: Molluscan resources and conservation 13 other hand, gelatinous egg masses appear to have no physical protection and one could reasonably hypothesise that these egg masses require chemical protection against both predation and disease. Ultimately, molluscan egg masses provide the opportunity to assess the relative importance of physical, chemical and behavioural adaptations to selective pressures in the marine environment.

Chapter 1. Introduction: Molluscan resources and conservation 14

Figure 1.3: The two main forms of molluscan egg masses; a) the gelatinous egg ribbons of Aplysia juliana (4x magnification) and b) the leathery egg capsules of an unidentified neogastropod (5x magnification) found washed up amongst beach debris.

Chapter 1. Introduction: Molluscan resources and conservation 15

1.4 Bioprospecting

1.4.1 Conservation and bioprospecting Bioprospecting can be described as the process of searching for pharmaceuticals in natural organisms. It has been suggested that the imperative to find novel natural chemicals before they disappear has made bioprospecting a politically respectable and fundable conservation tool

(Tangley, 1996). In a revolutionary proposal, the International Society of

Chemical Ecology suggested the financial coupling of chemical bioprospecting with conservation programs (Eisner and Meinwald, 1990).

There is a strong incentive for pharmaceutical companies to channel money towards the preservation of biodiversity and indeed several agreements have recently been negotiated to do just this (refer to Roberts, 1992; Sittenfeld and

Villers, 1993; Garson, 1996; Tangley, 1996; Aalbersberg 1997).

Another way in which bioprospecting can contribute towards conservation is through biodiversity research. A major component of conservation efforts involves the cataloging of biological resources and chemical screening itself can be considered as a form of inventory (Eisner, 1990). Wilson (1994) points out that chemical prospecting is heavily dependent on classification and is therefore best conducted in tandem with biodiversity surveys. The

International Union of Pure and Applied Chemistry (1996) recommends that bioprospectors provide support for surveys and inventories of existing fauna.

The search for new chemicals can also trigger the discovery of new species

(Garson, 1996) or rare species in new locations. The Manila Declaration

(1992) includes a code of ethics for foreign collectors of biological samples,

Chapter 1. Introduction: Molluscan resources and conservation 16 which suggests that collectors should “inform the host institute/appropriate organisation of new localities of rare/endangered species found”.

1.4.2 The ethics of bioprospecting There are a number of ethical issues surrounding chemical prospecting in the natural environment (ASTEC, 1998). Firstly, the collection of material for chemical and pharmacological studies is an extractive process and therefore could cause environmental impact. Secondly, the custodians of natural resources are stakeholders in the potential benefits arising from the commercial development of a natural product. There should be fair and equitable sharing of the results and benefits stemming from the utilisation and commercialisation of natural resources. Thirdly, intellectual property rights must be protected when traditional or other knowledge about the natural biota is shared with bioprospectors.

The ethical issues associated with bioprospecting have been addressed to some extent in several declarations, resolutions and other publications (i.e.

The International Convention on Biological Diversity, 1992; The Manila

Declaration, 1992; The Melaka Accord, 1994; Commonwealth Government of

Australia, 1994; International Union of Pure and Applied Chemistry, 1996a,b;

American Society of Pharmacognosy, 1997; The Commonwealth-State

Working Group on Access to Australia’s Biological Resources, 1997). These documents have primarily been developed to 1) facilitate access to biological resources, 2) ensure equitable benefit and 3) to protect the traditional

Chapter 1. Introduction: Molluscan resources and conservation 17 knowledge of indigenous people. There has been surprisingly little attention given to the potential environmental impacts of bioprospecting.

1.4.3 Is bioprospecting ecologically sustainable? Recently, there has been considerable concern that species have been exploited for pharmaceutical research in the ocean, with little regard to the potential impacts on their populations (Garson, 1996; 1997; Anderson, 1995;).

These concerns appear to be justified with reports such as 1000kg of the sea hare Dolabella auricularia being extracted to obtain 28mg of an anticancer peptide Dolastatin 10 (Pettit et al., 1987). Similarly, 5000 individuals of another sea hare Stylocheilus longicauda were extracted for 12g of

Aplysiatoxin (Kato and Scheuer, 1975). Although these two species are abundant with cosmopolitan distributions, these large collections could realistically represent a significant proportion of local populations. Without knowing the local abundance of the species, it is difficult to assess the true environmental impact of such large-scale collections.

However, reports of the large-scale collection of marine molluscs are actually quite uncommon. Of greater concern is the fact that the majority of papers published on marine natural products do not even report the amount of sample organism that has been collected. From a representative sample of papers on marine molluscs over half did not report the amount of sample collected (Figure 1.4; Appendix 1.1). This was found to be true of papers published in a wide range of chemistry and biology journals. Concern has also been raised over the fact that bioprospectors rarely provide any indication of

Chapter 1. Introduction: Molluscan resources and conservation 18 the abundance of the organisms they are collecting (Anderson, 1995). From a sample of 74 papers involving the isolation of natural products from marine molluscs, only 11 reported the abundance of the species collected (Figure

1.5; Appendix 1.1). Interestingly, a similarly low number of papers reported the amount and abundance of species collected for biochemistry, ecology and taxonomic studies on marine molluscs (Figure 1.4 & 1.5; Appendix 1.2).

Consequently, the concerns surrounding bioprospecting could extend into all fields of science that require the collection of organisms from the natural environment.

1.4.4 Requirements for sustainable bioprospecting The requirements for sustainable bioprospecting have not been previously addressed from a biological or ecological perspective. However, some biologically relevant recommendations have been made. In particular, the

International Union of Pure and Applied Chemistry (1996a) recommends that adequate provision be made for the preservation of rare or threatened species

“even to the extent of forbidding collections in some circumstances”. Similar recommendations were made at a Workshop in Kuala Lumpur (1996) in the guidelines for drafting a licence for access to biological resources. These recommendations are appropriate, however, they overlook the fact that the rarity of many organisms is simply not known.

The National Strategy for the Conservation of Australia’s Biological Diversity

(1997) outlines the need for non-threatening collection of biological resources.

Chapter 1. Introduction: Molluscan resources and conservation 19

80 not reported 70 sometimes reported 60 amount reported

Number of publications

0 Natural Biological Chemistry Biochemistry Experimental Natural History Products Biology Type of study Type of journal

Figure 1.4: The number of studies reporting the amount of specimens collected from a sample of 74 publications on molluscan natural products and 34 biological publications on molluscs. The proportion of papers that report the amount collected is listed for a variety of scientific journals. Papers that report the amount collected for some but not all species used in the study are listed as sometimes reported.

not reported 70 sometimes reported abundance reported 60

30 Number of publications

0 Natural Biological Chemistry Biochemistry Experimental Natural Products Biology History Type of study Type of journal Figure 1.5: The number of studies reporting the abundance of the species collected from a sample of 74 publications on molluscan natural products and 34 biological publications on molluscs. The proportion of papers that report the abundance of the species collected is listed for a variety of scientific journals. Papers that report the abundance of some but not all species used in the study are listed as sometimes reported.

Chapter 1. Introduction: Molluscan resources and conservation 20

This strategy states that we should ensure the “collection of genetic resources for research and development purposes does not adversely affect the viability or conservation status of the species or population being collected or of any component of its habitat”. The guidelines established in Kuala Lumpur (1996) also state that biological specimens should never be overcollected. However, neither of these documents outline the necessary requirements to help prevent the overcollection of specimens from the field.

There are two ways in which overcollection could take place during chemical prospecting. First, the collection of specimens could significantly reduce the local population size, leading to a decrease in genetic diversity, increased genetic drift or possibly local extinction. Many marine organisms have specialised habitats and small populations sizes and therefore could be susceptible to this type of overcollection. Consequently, research on natural history needs to be prioritised, to prevent the collection of vulnerable marine organisms.

Overcollection could also result from the collection of a greater number of specimens than is actually required for the proposed study. This type of overcollection may not have serious environmental impacts but it is unnecessarily wasteful and given our lack of knowledge about biological interactions, particularly in the marine environment, it can not be considered consistent with the objectives of conservation.

Chapter 1. Introduction: Molluscan resources and conservation 21

1.5 Molluscan conservation

1.5.1 The conservation status of marine molluscs The present state of knowledge on the conservation status of marine molluscs is poor. The criteria for assessing the conservation status of marine species are largely based on terrestrial situations and are generally not considered appropriate for marine taxa (Allison et al., 1998; Ponder, 1998; Chapman,

1998). There is currently little understanding of the rarity of marine invertebrates or threats to marine habitats. However, it is essential that an understanding of these factors is developed, in order to conserve marine biological diversity. Research aimed at improving the baseline data through systematic assessment of marine biota is necessary (Endangered Species

Advisory Committee (ESAC), 1996; Dept. Environment Sport and Territories

(DEST), 1997).

Molluscs were among the major invertebrate species identified by the

Endangered Species Advisory Committee (ESAC, 1996) as being of conservation concern in Australia. The species identified were primarily large bivalves and gastropods, at risk from over-exploitation for food and decoration. However, the species identified by the ESAC (1996) probably do not provide a comprehensive overview of the conservation status of marine molluscs because they primarily include commercially exploited species that have received considerable research attention. To date there is no reliable list of potentially threatened Australian marine molluscs (Ponder, 1998).

However, the Malacological Society of Australasia is currently preparing a list of marine molluscs with abbreviated life histories, low fecundity and/ or

Chapter 1. Introduction: Molluscan resources and conservation 22 restricted distributions (ESAC, 1996). These species are at the greatest potential risk from human impacts along the coast.

The temperate marine fauna of southern Australia is largely endemic (Norse,

1993; Edgar, 1997). In general however, the shallow water marine molluscs are widespread with planktonic larvae and are unlikely to become threatened

(Ponder, 1998; Ponder and Wells, 1998). A small fraction of species are believed to have restricted ranges and these are potentially vulnerable to habitat destruction and over-exploitation. Few species of mollusc are thought to have unusually restricted breeding sites (Ponder, 1998). However, some taxa with broad ranges are thought to be restricted in distribution by specific habitat requirements (Ponder, 1998). Extreme habitat specialists can be vulnerable if their required habitat is rare or threatened.

1.5.2 Habitat protection The identification and protection of important habitat and representative ecosystems is essential for conservation. Given the general shortage in time and funding, rapid recognition of threatened habitats containing large numbers of endangered and endemic species (the hot spots) could be essential for conservation. Wilson (1994, pg 320) has suggested “the primary tactic in conservation must be to locate the world’s hot spots and to protect the entire environment they contain”. This will require habitat mapping, community descriptions and inventories to provide an adequate assessment of conservation status (ESAC, 1996).

Chapter 1. Introduction: Molluscan resources and conservation 23

Only about 5.2% of Australia’s marine environment is currently protected in marine reserves (DEST, 1997). Much of this is within the Great Barrier Reef

Marine Park and large sections of Australia’s marine environment have few or no marine protected areas. The protection and conservation of intertidal habitat has been cited as being of paramount importance (ESAC, 1996). The state of the marine environment adjacent to highly populated areas can be locally poor and intertidal communities are most susceptible to habitat degradation associated with human impacts (DEST, 1997). Nevertheless, it is believed that Australia still has the opportunity to develop a truly representative system of protected marine ecosystems (Environment

Australia, 1998). The approach adopted by the Australian Government emphasises the use of scientific data in the identification of important areas and recognises that the necessary information is incomplete for many areas

(Environment Australia, 1998).

1.6 Structure of this study

The work described in this thesis has two primary aims, the first being to examine the role of natural products in defending molluscs egg masses and the second to explore the ways in which the discovery of new bioresources can contribute to the conservation of biological diversity. In order to combine bioprospecting with conservation it was necessary to ensure that the collection of specimens was conducted in an environmentally sustainable manner. There is currently little known about the diversity of intertidal molluscs on the Wollongong Coast, NSW, Australia and consequently, a species inventory has been provided in Chapter 2. This Chapter includes

Chapter 1. Introduction: Molluscan resources and conservation 24 information on the regional distribution and rarity of each species, as well as the intertidal breeding habitats used for the deposition of benthic egg masses.

An evaluation of the methods used for rapid biodiversity assessment in the intertidal zone is also provided in Chapter 2.

The role of chemical defence in the protection of benthic molluscan egg masses is explored in Chapters 3 and 4. Chapter 3 examines the role of chemical feeding deterrents using feeding trials against a range of ecologically relevant predators. The relative importance of physical and behavioural mechanisms for reducing predation is discussed. Chapter 3 also provides a preliminary examination of the macrofouling organisms associated with molluscan egg masses. Chapter 4 evaluates the antimicrobial properties of a wide range of benthic egg masses using two rapid screening methods. A preliminary examination of the microfauna associated with the egg masses is also included.

In Chapter 5, the compounds responsible for the antimicrobial activity in the egg masses of Dicathais orbita have been isolated using bioassay guided fractionation. The chemical structures of three antimicrobial metabolites have been elucidated using mass spectrometry and 1H NMR and their antimicrobial properties have been assessed against a range of human and marine pathogens. The results of preliminary molecular modelling studies are provided for one potential drug lead. In Chapter 6, the diversity of volatile organic compounds has been examined in a range of molluscan egg masses

Chapter 1. Introduction: Molluscan resources and conservation 25 using gas chromatography/ mass spectrometry. Some compounds potentially responsible for the observed antimicrobial activity have been identified.

Chapter 7 presents the general discussion and main conclusions from this study. Arguments are presented for the conservation of marine molluscs based on their resource potential. The value of using a biorational approach to drug discovery is also discussed and possible mechanisms are provided for ensuring future bioprospecting is environmentally sustainable. Finally, recommendations have been made for the conservation and management of intertidal molluscs along the Wollongong Coast, NSW, Australia.

NOTE

This online version of the thesis may have different page formatting and pagination from the paper copy held in the University of Wollongong Library.

UNIVERSITY OF WOLLONGONG

Chapter 2: Molluscan diversity 26

Chapter 2

Molluscan Diversity: Species inventories and habitat assessment.

2.1 Introduction

“ the diversity of life provides an insurance policy for a future in which change is inevitable.” E.A. Norse, 1993

2.1.1 Species diversity, species rarity and conservation implications Species richness (diversity) and the presence of rare species are the most frequently cited criteria for the selection of conservation reserves (Prendergast et al., 1993). Areas with a high diversity of species merit attention because they provide suitable conditions for the maintenance of large numbers of potentially competing species in sympatry (Norse, 1993). Highly biodiverse areas are often referred to as ‘hotspots’ (e.g. Wilson, 1994). The concept of ‘hotspots is primarily applied to the terrestrial environment. However, Norse (1993) suggests that it could be equally applied in the marine environment.

The immediate protection of ‘hotspots’ can be considered important for the conservation of biological diversity. However, the representation of all species in conservation planning can be problematic. Studies in the terrestrial environment have indicated that the occurrence of many rare (restricted range) species does not necessarily coincide with the location of hotspots (Prendergast et al., 1993; van Jaarsveld et al., 1998). However, Cunnett et al., (1994) suggest that the relationship between rarity and diversity is dependant on scale. These authors Chapter 2: Molluscan diversity 27

believe that areas of high diversity may still be the best way to protect rare species, as long as large areas are available for conservation. Little is known about the scale required to achieve effective conservation in the marine environment. However, it is clear that high species diversity should only be used as a measure of conservational importance within biogeographic regions and within, but not among, ecosystem types (Norse, 1993). In general, conservation planning is likely to be most effective at a regional level.

The protection of rare species is a central concern for many conservationists.

Rare species, which are usually regarded as those having low abundance and/or small ranges, can be more susceptible to environmental impacts, such as habitat destruction (Rabinowitz et al., 1986; Terborgh and Winter, 1990;

Gaston, 1994). An understanding of the rarity of species is also essential for the sustainable use of biological resources (e.g. bioprospecting, see Chapter 1;

Garson, 1996; 1997). An accurate assessment of species rarity is necessary to permit the selection of suitable species for chemical studies and help prevent overcollection.

There is a wide variety of viewpoints on the limits to what constitutes rarity in population biology (refer to Soulé, 1986b, Gaston, 1994). However, there is currently little understanding about the measures of rarity that are appropriate for marine organisms (Chapman, 1998). Some factors used to assess the rarity of terrestrial organisms may have applications in the marine environment. For example, species can be considered rare if they have a restricted geographic range, if they only occur in small numbers, or if they have specific habitat Chapter 2: Molluscan diversity 28

requirements, restricting them to a few specialised sites (Hubbell and Foster,

1986). Regardless of the definition that is used for rarity, the results will also be influenced by the spatial scale at which this definition is applied. Gaston (1993) suggests that the concept of rarity can be applied at almost any spatial scale, although it has been most extensively studied at the regional level. A cautious approach to marine conservation should include the protection of regionally rare species, at least until we have a greater understanding of the genetic structure of the populations across a larger geographic scale.

Species diversity and rarity can also be influenced by the immigration of individuals into an area outside their normal range. Shimida and Wilson (1985) argue that the flow of individuals into an area is one of the major biological determinants of species richness. A species that has recently immigrated from population centres outside the study area will typically be classified as rare (e.g.

Hubbell and Foster, 1986) primarily because they occur at low abundances.

The concept of immigrants as an influence on species rarity and diversity has relevance to the assessment of species in the intertidal zone because some species could infrequently migrate in from subtidal habitats.

Underwood (1994) has commented that in general, we lack information about the rarer species along the Australian coast. For example, no rare molluscs have been previously recorded along the Illawarra Coast (Owen, 1978;

Miskiewicz, and Lock, 1991; Marine Pollution Research (MPR), 1992; Marine

Pollution Research (MPR), 1995; Minchinton, 1996; Waters, 1997). However, rare species are likely to be under-represented in these studies because the Chapter 2: Molluscan diversity 29

methods used to sample intertidal reefs typically provide little information about rare species (Underwood, 1981). Furthermore, spatial and temporal replication has been extremely limited in the previous studies along the Illawarra Coast

(Owen, 1978; Miskiewicz, and Lock, 1991; MPR, 1992; Minchinton, 1996;

Waters, 1997). However, the abundance of many marine invertebrates is highly variable in space and time (Underwood and Chapman, 1997).

2.1.2 Intertidal reefs and the determinants of species diversity The effective conservation of biological diversity is also dependent on our understanding of the interactions between organisms and their habitat. The rarity of species can depend on the availability of suitable habitat and in particular, the frequency of suitable microsites for successful reproduction (refer to Hubbell and Foster, 1986). In general, breeding and nursery grounds are considered to have high ecological importance, which merits high priority for their protection (Norse, 1993).

Smith (1972), reports that many molluscs deposit egg masses on the underside of boulders. Consequently, the availability of suitable boulders might be one factor influencing the diversity of molluscs on intertidal reefs. It has also been suggested that molluscs will deposit egg masses in an environment similar to that required by the adult for its own existence (Allan, 1941; Spight, 1977).

Therefore, information on the breeding sites used by intertidal molluscs could provide general information about the habitat requirements of some species.

Overall, there is a great need for more scientific data on how organisms interact with the biophysical environment (Fairweather, 1989). Chapter 2: Molluscan diversity 30

A number of quantitative studies have been conducted on Australian intertidal rocky shores (refer to Underwood, 1994) but these studies all focus on conspicuous intertidal organisms and their patterns of distribution. There are no studies comparing the diversity of a group of organisms across a range of intertidal reefs. This type of study would not only facilitate the identification of sites containing high species diversity but could also be used to investigate environmental factors influencing intertidal diversity.

One factor that could potentially influence species diversity on intertidal reefs is exposure to wave action. Observations suggest that many intertidal organisms prefer some degree of shelter from the swell (refer to Allen, 1975; Bennet,

1992). Nevertheless, Underwood (1981) found no correlation between diversity and the degree of wave exposure, in a study conducted along a gradient of wave exposure on a single rock-platform. This study concentrated on common species and some organisms were not sampled due to rarity, rapid mobility or taxonomic uncertainty. More importantly, the study site did not represent the full range of wave exposure that can be found on the N.S.W. coast (i.e. the extremes of sheltered and exposed conditions were lacking on this rocky shore). In a similar vein, Owen (1978) attempted to assess the difference in intertidal diversity between exposed (south-east facing) and sheltered (north- west facing) aspects at several sites along the Illawarra Coast, N.S.W. This study also found no difference in species richness with wave exposure.

However, it was noted that the apparently sheltered aspects received almost as much wave action as those sites directly exposed to the severity of wave action. Chapter 2: Molluscan diversity 31

More quantitative studies comparing sheltered and exposed shores in Australia would be useful (Underwood, 1994).

The availability of microhabitats may also affect species diversity on intertidal reefs (see McGuiness, 1984b). The Habitat Diversity hypothesis (Connor and

McCoy, 1979) predicts that a greater diversity of species can occur where a greater variety of habitats exist. This hypothesis has been experimentally tested, by manipulating the availability of microhabitats on intertidal boulders

(McGuiness and Underwood, 1986). It was found that the diversity of habitats did influence the colonisation of boulders by sessile and grazing invertebrates.

However, the patterns of response to the availability of microhabitats varied among species and the authors concluded that there was a need for further studies on the effects of habitat structure on intertidal communities.

The influence of habitat diversity on intertidal species diversity has not been addressed on a large scale i.e. across different intertidal reefs of varying habitat complexity. However, the structure of intertidal habitats can vary markedly. For example, the presence of boulder fields, rock pools, rocky outcrops, caves and crevices will influence the area and diversity of habitat on an intertidal reef

(Bennet, 1992; pers. obs.). The quality of intertidal habitat can also be influenced by the slope of the reef (Bennett, 1992). Gently sloping reefs provide a large area of horizontal intertidal habitat, whereas the intertidal area is vertical and more limited on rock platforms that drop abruptly into the ocean.

Observations suggest that all of these habitat features could contribute to intertidal diversity (Allen, 1975; Bennett, 1992). Chapter 2: Molluscan diversity 32

Habitat structure is generally easier to assess than biological diversity or ecosystem functions. Consequently, the establishment of links between certain habitat types and the diversity of intertidal organisms could greatly improve our ability to make sensible management decisions along the Australian coast.

Analysis of biological and geophysical data could also assist in the rapid identification of hotspots of biological diversity and this may be useful for the establishment of a comprehensive, representative and adequate reserve system.

2.1.3 Species inventories and rapid biodiversity assessment Species lists can be considered important data sets for conservation and resource management (Oliver and Beattie, 1993; Soulé and Kohm, 1989; Stork,

1994). Janzen (1993) suggests that the compilation of biodiversity inventories, is the first step towards sustainability. In particular, bioprospecting is facilitated by knowledge of the existing species and their distributions in space (Wilson,

1994; International Union of Pure and Applied Chemistry, 1996). However, species lists can only be considered useful if they provide an accurate representation of the species diversity and their abundance in the region.

Quantitative measurements of biodiversity are necessarily based on inventory and systematics. However, there is a worldwide shortage of taxonomists and an increasing need for accurate species identification (Norse, 1993; Wilson, 1994).

Consequently, methods for the rapid assessment of biodiversity have been recently tested, which utilise non-specialist parataxonomists or biodiversity Chapter 2: Molluscan diversity 33

technicians (Gamez, 1991; Oliver and Beattie, 1993; 1994). Oliver and Beattie

(1993; 1994) have demonstrated that for some taxa, rapid biodiversity assessment can produce results close to the formal taxonomic estimates.

Species inventories are often conducted for the purpose of Environmental

Impact Assessment (EIA) and local management plans. There has been much criticism about the use of science in EIA (Buckley, 1989; Fairweather, 1989;

Benkendorff, in press). In particular, the ecological surveys have been criticised for using inappropriate methodologies, which do not incorporate the most recent ideas and techniques published in the scientific literature (Peterson, 1993;

Fairweather, 1994). A variety of study approaches have been discussed in the literature (Bernstein and Zalinski, 1983; Lincoln-Smith, 1991; Underwood, 1991;

1992; 1998) and in general it has been suggested that the most appropriate ecological studies involve repeated sampling to account for spatial and temporal variation. However, most studies conducted for environmental impact assessment are limited by restrictions in time and cost (Fairweather, 1989;

Warwick, 1993; Fairweather, 1994). Consequently, there is a need to develop rapid survey methods that provide an accurate assessment of the environment, within the constraints imposed on ecological consultants for the purpose of EIA.

Chapter 2: Molluscan diversity 34

2.1.4 Molluscs of the Illawarra Coast The Illawarra Coast extends along the south-east of New South Wales,

Australia, from Port Hacking to the Shoalhaven River (refer to Figure 2.1a). The research sites used in this study are primarily located on the Wollongong coast

(Figure 2.1b). For the purpose of this study, the Wollongong region of the

Illawarra Coast is specified as the area extending 30km north and south from

Wollongong, NSW (Figure 2.1a). A high diversity of intertidal fauna is expected along the Illawarra Coast, because it contains a mix of tropical and southern

Australian biota (e.g. Allan, 1962).

The predominant swell along the Illawarra Coast is from the south-east and therefore, north-facing reefs are the most protected from strong wave action.

Two large headlands with sheltered intertidal reefs are found in the Wollongong region; Bass Point and Bellambi Point (Figure 2.1). A large number of wave exposed reefs with a range of habitat complexity can also be found on the

Wollongong coast. Consequently, the intertidal reefs in this region provide an opportunity to investigate the relationship between species richness and the geophysical environment.

There has been little effort to comprehensively assess the species diversity or conservation status of marine molluscs along the Illawarra Coast. However, a number of previous studies have sampled the intertidal invertebrates in the region (Owen, 1978; Miskiewicz, and Lock, 1991; MPR, 1992; MPR, 1995;

Minchinton, 1996; Waters, 1997). These are all small-scale studies involving sampling at a few sites over a short period of time. The diversity of molluscs Chapter 2: Molluscan diversity 35

! Sydney N

" Towra Point

! Port Hacking

N.S.W

SEA TASMAN

! Wollongong Figure 2.1b

" Lake Illawarra

Shoalhaven Heads Nowra !

St Georges Basin "

Figure 2.1a: The Illawarra Coast, N.S.W., Australia from Port Hacking, Sydney to the Shoalhaven Heads, Nowra. " indicates the location of some estuarine sampling sites. Chapter 2: Molluscan diversity 36

$" Coalcliff $ Scarborough $" Wombarra $" Coledale $" Austinmer

$" Bulli

$" Bellambi BELLAMBI POINT

$" Towradgi

Wollongong ! & Puckeys Creek Port Kembla North Wollongong $" Wollongong " Channel 0 34 30' Lake Wollongong $ Flagstaff Hill Illawarra Harbour " Windang

Shellharbour ! $ North Shellharbour $ South Shellharbour $% Bass BASS POINT Point

151 0 50'

Figure 2.1b: The Wollongong region of the N.S.W. coast, showing the intertidal sampling sites: ! natural reefs; " artificial habitats; # estuarine habitats. . Chapter 2: Molluscan diversity 37

recorded in these studies is low compared to the number of species known to occur on N.S.W. shores (Figure 2.2; refer to Iredale and McMichael, 1962;

Bennett, 1992; Wilson, 1993; Edgar, 1997). Indeed, the species diversity recorded in the previous studies along the Illawarra Coast is likely to be seriously underestimated. For example, only 13 species of prosobranchs were recorded at Shellharbour by Marine Pollution Research (1995), whereas 11 species from just one family of prosobranch gastropods (Cypraea) have been identified at Shellharbour from museum records (Tarrant, 1980).

Museums and numerous private collectors hold large collections of shells with potentially valuable information on the distribution of molluscs (Underwood,

1974; Norse, 1993; Nielsen and West, 1994; Ponder, 1997). The Australian

Museum holds a large collection of shells from Shellharbour on the Illawarra

Coast. However, only 5-10% of this collection has so far been databased (Ian

Loch, pers. comm.). Nevertheless, with 47 species, this database provides the most comprehensive current record of shelled molluscs on the Illawarra Coast

(Figure 2.2). By comparison, soft-bodied molluscs generally do not preserve well and are therefore under-represented in museum collections. This means that there are few historical records on the distribution of these species. It is therefore of concern that only one species of soft-bodied mollusc has been previously recorded on the Illawarra Coast (Figure 2.2; MPR, 1995). Chapter 2: Molluscan diversity 38

120

100 Cephalopods Bivalves Chitons

80 Opisthobranchs Pulmonates Prosobranchs 60 Number of species 40

0 Bennett, Edgar, 1997 Owen, 1978 Miskiewicz MPR, 1992 MPR, 1995 Minchinton, Waters, Australian Tarrant, 1992 and Lock, 1996 1997 Museum, 1980 1991 1997

Common molluscs Molluscs recorded in previous studies on the Illawarra Coast Shell collections from N.S.W. from Shellharbour

Figure 2.2: The number of species of common molluscs recorded in New South Wales (Bennett, 1992; Edgar, 1997) compared to the number of molluscs recorded in previous studies along the Illawarra Coast (Miskiwiicz and Lock, 1991; Marine Pollution Research (MPR), 1992; 1995; Minchinton, 1996; Waters, 1997). The species regarded as common in the field guides by Bennett (1992) and Edgar (1997) are regularly observed at most intertidal reefs along the the N.S.W. coast. Records of shell collections from the Illawarra region are also included (Australian Museum, 1997: ~5-10% of shell collection from Shellharbour; Tarrant, 1980: Cypraea from Shellharbour).

Chapter 2: Molluscan diversity 39

2.2 Objectives

The primary objectives of this aspect of the research project were to assess the distribution and abundance of intertidal molluscs along the Wollongong coast,

N.S.W, Australia and to identify species suitable for the collection of their egg masses. Abundant molluscs found depositing egg masses in estuarine environments along the Illawarra Coast were also recorded. The breeding sites used by molluscs for the deposition of benthic egg masses was investigated on intertidal reefs. Preliminary observations were made on the physical features of each intertidal reef, in particular the degree of wave exposure, the slope of the reef and the habitat complexity. These environmental features could then be correlated with molluscan diversity. Spatial and temporal variation in the molluscan fauna was compared across different intertidal reefs to determine the amount of survey effort required to gain a reasonable estimate of species diversity. Ultimately, this information should contribute towards the development of rapid and sound intertidal survey methods for the purposes of environmental impact assessment and resource management, including bioprospecting.

2.3 Methods

2.3.1 Study sites Surveys of the molluscan fauna were conducted at a number of intertidal habitats along the Illawarra coast. These included thirteen intertidal reefs, eight artificial swimming pools and one artificial channel (Figure 2.1b). A summary of the physical features for each intertidal reef are provided in Table 2.1. These include the degree of exposure to swell, the slope of the reef, the type of rock, the number of microhabitats and the aerial extent of boulders. The following Chapter 2: Molluscan diversity 40

microhabitats were identified; flat rock platforms, boulders, rocky outcrops, crevices, caves, patches of sand, rock pools and shallow water-retaining hollows (Table 2.1).

The majority of intertidal reefs in the Wollongong region are exposed to heavy swell (Table 2.1). All of the exposed reefs along the Wollongong Coast are periodically subject to natural impacts from extreme swell conditions and sand inundation. These exposed reefs vary significantly in degree of habitat complexity, as judged from the area of boulders and variety of other habitat features (Table 2.1). Most of the exposed reefs are dominated by flat rock platforms with a large supralittoral belt above the high tide mark (e.g. Figure

2.3a). The intertidal zone is narrow and primarily vertical on these wave-cut rock platforms. Six representative wave-exposed rock platforms were selected for regular sampling (Coalcliff, Scarborough, Wombarra, Coledale, Austinmer and

Flagstaff Hill; Table 2.1).

Gently sloping, wave-exposed intertidal reefs (e.g. Figure 2.3b) are much less common on the Wollongong Coast. Two sites, (Bulli and Towradgi) were selected for regular sampling and compared to the steep rock platforms, these reefs both have a high degree of habitat complexity (Table 2.1).

Sheltered intertidal reefs can be found on the northern side of Bellambi Point and Bass Point. One site was selected for sampling on the northern side of

Bellambi Point (Bellambi) and three sites were selected on the northern side of

Bass Point (North Shellharbour, South Shellharbour and Bass Point; Table 2.1). Chapter 2: Molluscan diversity 41

These sheltered sites rarely experience swell greater than one metre and all are gently sloping with a high degree of habitat complexity (Table 2.1; e.g. Figure

2.3c).

Red Point (Port Kembla) and Flagstaff Point (Wollongong) also provide potential sheltered sites along the Wollongong Coast (Figure 2.1). However, the northern side of these two headlands have been converted into boatharbours. One moderately exposed intertidal reef was found on the north-eastern side of

Wollongong Harbour (North Wollongong; Table 2.1). This site can experience large swell (ie. waves >1m). However, it is sheltered from the full force of southerly and south-easterly swell.

Artificial habitats were also sampled at a number of sites along the Wollongong

Coast (Table 2.1). With the exception of Wollongong Channel, these are all human-made swimming pools, which have been constructed on rock platforms.

The artificial swimming pools have a low habitat complexity with only vertical and horizontal substrata provided by the walls and the bottom of the pools

(Figure 2.4a). The bottom of most of the pools is cement but they frequently accumulate sand, which may provide an estuarine-like habitat. Wollongong

Channel is a naturally formed boulder-filled channel, which has been blocked off at the eastern end by a cement wall. Like the pools, this channel is sheltered from most wave action but it is gently sloping and has a greater degree of habitat complexity as a result of the presence of many boulders.

Chapter 2: Molluscan diversity 42

Figure 2.3: The three main types of intertidal reefs on the Wollongong coast, N.S.W., Australia; a) a typical wave exposed rock platform at Scarborough, with a large area of supralittoral habitat and a narrow intertidal zone that drops vertically into the ocean; b) a gently sloping, wave exposed reef at Towradgi with a large area of intertidal habitat with a high degree of complexity; c) a gently sloping, north facing reef at Shellharbour, with a large area of complex intertidal habitat protected from strong swell. Chapter 2: Molluscan diversity 43

Figure 2.4: ‘Estuarine’ habitats on the Wollongong coast, N.S.W. Australia; a) a typical artificial swimming pool at Bellambi. The pool has been drained for cleaning by Wollongong City Council; b) a natural estuary at Puckeys Creek, Fairy Meadow. Chapter 2: Molluscan diversity 44

Preliminary observations of the common molluscs found depositing egg masses in several natural estuarine environments along the N.S.W. coast were also recorded. These were taken from Towra Point, which is just south of Sydney, the entrance to Puckeys Creek, Fairy Meadow (Figure 2.4b) and Lake Illawarra in the Wollongong region, as well as St Georges Basin, which lies further south on the N.S.W. Coast (Figure 2.1).

2.3.2 Surveys The number of surveys conducted at each study site is summarised in Table

2.1. These vary across the different sites, mostly due to the difficulty of accessing the site. The sampling dates are recorded in Appendix 2.1 and 2.2.

The artificial pools were sampled on the same days as the adjacent natural reef, except for Bellambi Pool, which was only sampled on six occasions (Appendix

2.2).

Data is presented for a single inventory of each site, in addition to the accumulated sampling records. The single inventories have been used for across site comparisons, whereas the accumulated sampling records enabled an assessment of temporal variation and provided a more comprehensive list of the intertidal molluscan fauna of the Wollongong region. Sampling was conducted over a three year period, from Autumn 1995 to Autumn 1998. The inventories of each site were conducted after sufficient familiarity with the range of species found in the region, had been developed. All inventories were done on low Spring tides (0.1-0.2m) from late 1997- early 1998. Chapter 2: Molluscan diversity 45

Table 2.1: Summary of the natural and artificial intertidal habitats surveyed at 14 sites along the Illawarra Coast, NSW, Australia. Site No. Wave Rock type Slope Area Natural habitat features Artificial habitat Surveys exposure containin g boulders Austinmer 16 Highly Sandstone Steep < 20m2 Large rock platform with deep crevices Two large swimming exposed pools and one shallow pool with sandy bottom Bass Point 3 Moderately Basalt Moderate- 50-100m2 Rock platform with shallow boulder filled - exposed steep hollows, caves and crevices Bellambi 19 Sheltered Sandstone Gentle > 100m2 Large boulder filled hollows and rock platform One large and one with caves and crevices small swimming pool Bulli 19 Highly Sandstone Gentle- 50-100m2 Rock platform with boulder filled hollows and One large and one exposed moderate crevices, deep rock pools, sandy patches, small swimming pool caves and rocky outcrops Coalcliff 10 Highly Sandstone Moderate- < 50m2 Large rock platform with some boulder filled One large and one exposed steep hollows, shallow pools, caves crevices small swimming pool Coledale 9 Highly Sandstone Gentle- < 20m2 Large rock platform with shallow pools and One large and one exposed steep crevices and small patch of submerged small swimming pool boulders Flagstaff Hill 17 Highly Sandstone Steep < 20m2 Rock platform with deep pools, crevices, One pool but not exposed caves and a small reef flat sampled North 2 Sheltered Basalt Gentle > 50m2 Rock platform interspersed with shallow - Shellharbour boulder filled hollows and crevices North 14 Moderately Sandstone Moderate- < 50m2 Rock platform interspersed with boulder filled Two swimming pools Wollongong exposed gentle crevices, rocky outcrops with caves Scarborough 11 Highly Sandstone Steep < 2m2 Rock platform with one large pool and several - exposed crevices South 15 Sheltered Basalt Gentle >> 100 Rock platform interspersed with large boulder - Shellharbour m2 filled hollows, sandy patches, rocky outcrops, caves, crevices Towradgi 20 Highly Sandstone Gentle > 100m2 Large boulder field with small rock platform, One large and one exposed caves, rocky outcrops and sandy patches small swimming pool Wombarra 15 Highly Sandstone Moderate- < 50m2 Large rock platform and off-shore reef with One large and one exposed steep caves, crevices and boulder filled hollows small swimming pool Wollongong 15 Sheltered Sandstone Gentle < 50m2 - Boulder filled channel channel Chapter 2: Molluscan diversity 46

Sampling involved searching the intertidal area for molluscs (>5mm) and their egg masses during the three to four hour period over low tide. No quadrats or transects were used but rather the entire range of habitats were searched at all levels of the shore. In particular, the undersides of boulders were investigated, both in dry regions and in water retaining hollows. Other rock surfaces, such as vertical rock faces, caves and crevices, were investigated as far as possible.

Notes were taken on the type of substratum used by molluscs to attach egg masses.

Some additional records have been made of shells and egg capsules washed up amongst the beach debris. These surveys should not be taken as a comprehensive species list of the molluscan fauna in the Wollongong area.

Many species are not likely to have been recorded in these surveys. In particular, free spawning molluscs, such as bivalves, chitons and limpets, as well as, small, cryptic and ephemeral species, are likely to have been overlooked.

The molluscs have been identified according to Wilson (1993), Beesley et al.,

(1998) and Edgar (1997), with the assistance of the Australian Museum. Where there are discrepancies in the species names of common species between the publications the nomenclature of Wilson (1993) was used. Voucher specimens of most of the shelled molluscs and some of the egg masses have been lodged in the Department of Biological Sciences, University of Wollongong. Soft-bodied molluscs and gelatinous egg masses do not preserve well and are better represented by colour photographs. Consequently, photographs have been Chapter 2: Molluscan diversity 47

taken of all the egg masses, as well as the soft-bodied molluscs and other species deemed to rare to warrant preservation.

The abundance of species has not been quantified (except for rare species) but rather a subjective ranking has been used based on experience. There is no formal ranking system for assessing the abundance of molluscs available in the literature. Consequently, abundance has been estimated at each site from observations taken over the entire reef during three years of field work. Species were regarded as abundant when more than 200 individuals were consistently recorded at the site. Species were classified as common when greater than 30 individuals were observed on more than half of the trips to a particular site.

Uncommon species were constantly recorded in low numbers (< 30 individuals) at a particular site, or were recorded in moderate numbers (10-50 individuals) on only one or two occasions. Species were regarded as rare when less than 10 individuals were recorded at the site over the entire three year period. The species abundance at each study site is recorded in Appendix 2.1.

The regional rarity of each species has been assigned according to their distribution in the Wollongong intertidal region and their abundance at each site.

Species that were only found on sheltered (north facing) reefs were considered to have restricted distributions in the region. Whereas, species that occur on wave exposed rock platforms were considered to have wide available habitat.

Species were classified as regionally rare if they were rare at every site where they were observed or if they were uncommon with restricted distributions.

Species that were common with restricted distributions or uncommon with wide Chapter 2: Molluscan diversity 48

distributions were considered to be regionally uncommon. Common or abundant species with wide available habitat were regarded as regionally common.

2.3.3 Habitat quality Preliminary observations have been made on the physical environment at each intertidal site (Table 2.1). Each site has been ranked according to three physical features; wave exposure, slope and habitat complexity (Table 2.2). A ranking system was devised for the variation in each geophysical feature ranging from one to five (see below). These rankings were correlated to the species diversity

(effectively species richness) of molluscs found at each site. The species diversity was taken from a single inventory, due to variation in the total number of surveys conducted at each site. The species inventories were all conducted on a low spring tide and approximately four hours was spent looking for molluscs at each site.

Slope refers to the degree in which the intertidal rock shelf drops into the ocean.

The slope of the intertidal reefs varies from gentle (0-200C), through moderate

(20-500C), to steep (50-900C) along the Wollongong Coast (Table 2.1). Steeply sloping reefs have a large supralittoral belt (or splash zone) and a narrow belt of primarily vertical habitat, in the littoral zone (the true intertidal area). However, the slope is not always consistent across the reef. Therefore the rankings for slope are estimated from an average across the reef. Reefs with steep (i.e. primarily vertical) drop offs were ranked at one, whereas gently sloping reefs were ranked at five (Table 2.2). Chapter 2: Molluscan diversity 49

Habitat complexity has been estimated according to the variety of potential microhabitats present and the overall area containing boulders (Table 2.1).

Sites with large areas of boulders (>100m2) and several other microhabitats were ranked at five. Sites with no significant patches of boulders and few of the other habitat features were ranked at one (Table 2.2). Under these definitions habitat complexity could be confounded with the size of the reef or total area of habitat.

Exposure to wave action was estimated according to the aspect of the reef and the degree of protection from adjacent headlands. Sites which are facing north are considered more sheltered due to the predominate south-easterly swell on the Illawarra Coast. The approximate distance from the end of the point was also taken into consideration. Sites that are greater than 100m from the point with a completely northerly aspect were considered to be sheltered and were ranked at five. Moderately exposed sites are less than 100m from the point and/or facing north-east and these were ranked at four or three. Sites with >

1800 outlook across the ocean were considered to be fully wave exposed and these sites were ranked at one (Table 2.2).

The ease of accessing and sampling the intertidal area at each site has also been taken into consideration. These factors were estimated subjectively from my experience of working at a range of intertidal reefs over three years. The ease of access to the intertidal area at a site influences the number of days and types of weather conditions that are suitable for conducting intertidal studies. In Chapter 2: Molluscan diversity 50

general the intertidal area is easier to access at sites that are gently sloping and/or sheltered. However, ease of access should not influence the number of species recorded in the species inventories during this study because the inventories were all conducted on low spring tides during calm sea conditions.

The ease of sampling the intertidal zone is primarily influenced by the habitat complexity and the slope of the intertidal reef. It is more difficult to comprehensively sample sites with a high degree of habitat complexity and in particular, large areas of loose boulders are time consuming to examine.

Vertical rock surfaces on steep rock platforms are also difficult to sample because they are difficult to access even during calm sea conditions. All the gently sloping reefs selected for sampling in this study have a high habitat complexity and the steep rock platforms tend to have a lower habitat complexity

(refer to Tables 2.1; 2.2). Therefore, these factors that could influence the ease of sampling in the intertidal zone tended to balanced out across the different study sites such that ease of sampling appears to be similar for all study sites.

Consequently, ease of sampling is unlikely to significantly influence the diversity of molluscs recorded in this study.

It should be noted that all of the rankings provided in Table 2.2 are subjective.

They are essentially based on my familiarity with each site, from repeated sampling on a range of tide and swell conditions. For the purposes of this study it was not deemed appropriate to take quantitative measurements of the physical environment during the limited time available at low tide, in addition to searching for egg masses and conducting surveys of the molluscan fauna. Chapter 2: Molluscan diversity 51

Rather, the relationship between molluscan diversity and the physical environment has been included as a preliminary study to indicate areas suitable for future research. Accurate measurements of habitat complexity and slope would require replicated transects across the entire reef. These measurements should be taken on calm low Spring tides (0.2-0.0m), because some rock platforms contain small patches of boulders that can only be accessed in these conditions. Accurate measurements of wave exposure would require calculations on a number of days, encompassing the full range of weather conditions, to obtain averages that can be compared across sites.

2.3.4 Statistical analysis Statistical analyses were performed using the JMP statistical package. A

Pearsons Chi squared test for heterogeneity was performed to determine the likelihood that species were recorded in previous studies along the Illawarra

Coast based on their rarity, as determined along the Wollongong coast in this study.

Correlations were performed to test the relationship between the number of species and the habitat quality rankings in Table 2.2. Each feature was assessed separately but there is the potential that these features interact to influence species diversity. However, the physical features were not analysed in combination because they may not be independent.

Chapter 2: Molluscan diversity 52

Table 2.2: Habitat rankings for thirteen intertidal reefs along Wollongong Coast. Each site has been ranked for three physical features; wave exposure, slope and habitat complexity. The rankings were assigned completely independent of the diversity data. A ranking of 1 indicates the site is highly exposed to wave action, steeply sloping and with low habitat complexity. Whereas, a ranking of 5 indicates the site is sheltered from strong wave action, gently sloping and with a high degree of habitat complexity.

Site Wave Slope Habitat exposure complexity Austinmer 1 2 2 Bass Point 4 4 5 Bellambi reef 5 5 5 Bulli 1 4 4 Coalcliff 1 3 3 Coledale 1 3 3 Flagstaff Hill 1 1 3 North Shellharbour 5 5 5 North Wollongong 3 4 4 Scarborough 1 1 1 South Shellharbour 5 5 5 Towradgi 1 5 5 Wombarra 1 2 3

Chapter 2: Molluscan diversity 53

2.4 Results

2.4.1 Species list of intertidal molluscs in the Wollongong Region A total of 176 molluscs were found on the intertidal reefs along the Wollongong

Coast (Table 2.3). Fifteen of these molluscs are represented by unidentified juveniles or egg masses, which could also have been recorded as adults.

Consequently, only 161 of the molluscs recorded in this study can be unambiguously considered as separate species. In total, 150 unambiguously distinct species were recorded live in the intertidal area and a further 11 were found washed up on beaches (Table 2.3). The majority of species found live in the intertidal area were regionally rare (66%) and only 33 species of molluscs may be regarded as common in the Wollongong intertidal area (Table 2.3). The species list for each site (listed in Table 2.1) is presented in Appendix 2.1.

Overall, 55.7% of the molluscs listed in Table 2.3 were recorded amongst beach debris and 17 of these species were not recorded live (Table 2.3). The shells from ten additional species were found more frequently than the live individuals

(Table 2.3). The shells from ten other species were recorded less frequently than the live specimens in the intertidal area (Table 2.3). Only two soft-bodied molluscs, both Cephalopods (Sepioteuthis australis and Sepia sp.), were found washed up on the beach (Table 2.3).

The egg masses deposited by 59 distinct species of molluscs have been recorded from the intertidal area along the Wollongong Coast. The species responsible for depositing 12 of these egg masses have not yet been identified and seven of these were egg capsules found washed up on the beach (Table Chapter 2: Molluscan diversity 54

2.3). The egg masses from the remaining species were identified by witnessing the individuals depositing in the field or from deposition in aquaria. In total, 24 species were found to deposit egg masses in aquaria (Table 2.3). Most of the

Opisthobranchs that were held in aquaria deposited egg ribbons within two days after capture from the field. The egg capsules from one prosobranch (Nassarius jonasii) were not observed in the field. These egg capsules were found to be small and cryptic when deposited in the aquarium. Sixty one percent of the egg masses found were deposited by species that were regionally rare. Only ten regionally common species and a further 10 uncommon species were found depositing egg masses in the Wollongong intertidal reefs.

The egg masses from six further species were found in natural estuarine habitats (Table 2.4). All of these species appear to be very common, although the species responsible for depositing one of these egg masses has not been identified. This species is most likely to be pulmonate based on the habitat (i.e. high intertidal mud flats) and the appearance of the egg ribbons. The egg mass of Conuber cf. sordidus were observed much more frequently than the adults.

None of these species were recorded on intertidal reefs in the region (Table

2.3).

The egg ribbons of an unidentified polychaete worm were also observed at

Lake Illawarra in large numbers. The egg masses from two different polychaetes were found in the artificial pool at Bulli. These were both rare and were only observed when the pool was drained for cleaning. The polychaete Chapter 2: Molluscan diversity 55

Table 2.3: Species list of intertidal molluscs recorded from 13 natural reefs, eight artificial pools and one artificial channel in the Wollongong Region during 1995-1998. The rarity of each species has been included for both specimens that were recorded live in the intertidal area and beach washed specimens found in the Wollongong region (see text for a definition of rarity). All species found depositing egg masses have been recorded and several unidentified egg masses have been listed. F indicates the eggs were observed in the field and A is used to designate species that deposited eggs in aquaria. Previous studies refer to the number of studies on the Illawarra Coast that have previously recorded the listed species. The previous studies include:- Owen, 1978; Tarrant, 1980; Miskiewicz, and Lock, 1991; MPR, 1992; MPR, 1995; Minchinton, 1996; Australian Museum, 1997; Waters, 1997. Family Species Regional Egg Beach Number of rarity masses wash previous studies CLASS GASTROPODA; SUBCLASS EOGASTROPODA Order Pattellogastropoda Patellidae Patella peronii Common - Common 1 Patellidae Patella chapmani Rare - Common 0 Patellidae Cellana tramoserica Common - Common 5 Acmaeidae Notoacmea petterdi Common - Common 4 Acmaeidae Patelloida alticostata Common - Common 3 Acmaeidae Patelloida cf. mufria Uncommon - Uncommon 1 SUBCLASS ORTHOGASTROPODA Architectonicidae Philippia lutea Rare - - 0 Superorder Vetigastropoda Haliotidae Haliotis ruber Rare - Rare 2 Haliotidae Haliotis coccoradiata Rare - Rare 0 Haliotidae Haliotis cf. melculus - - Rare 0 Fissurellidae Scutus antipodes Common - Common 3 Fissurellidae Amblychilepas nigrita Uncommon - Rare 0 Fissurellidae Diodora lineata Uncommon - Rare 1 Fissurellidae Tugali sp. Rare - Rare 0 Fissurellidae Montfortula rugosa Common - Common 5 Trochidae Austrocohlea constricta Common - Common 6 Trochidae Cantharidella picturata Common - Uncommon 1 Trochidae Clanculus brunneus Rare - Rare 0 Trochidae Clanculus clangus Rare - Rare 1 Trochidae Clanculus floridus Rare - Rare 0 Trochidae Granata imbricata Uncommon - Uncommon 0 Trochidae Herpetopoma aspersa Rare - Rare 1 Trochidae Phasianotrochus eximius Common - Common 0 Trochidae Phasianotrochus sp. Rare - Rare 0 Trochidae Stomatella impertusa Rare - - 0 Trochidae Tallorbis roseolus Rare - Uncommon 0 Turbinidae Astralium rhodostomum Rare - Rare 0 Turbinidae Astralium squamiferum Rare - Rare 0 Turbinidae Astralium tentoriformis Uncommon - Uncommon 2 Turbinidae Turbo torquatus Rare - Uncommon 3 Turbinidae Turbo undulatus Common - Common 5 Turbinidae Phasianella sp. Rare - Rare 0 Superorder Neritopsina Neritidae Nerita atramentosa Common F, A Common 6 Superorder Caenogastropoda; Order Sorbeoconcha Batillariidae Velacumantus australis Rare - Rare 0 Dialidae Diala sp. Rare - Rare 0 Cerithiidae Cacozeliana granaria Uncommon - Uncommon 1 Chapter 2: Molluscan diversity 56

Family Species Regional Egg Beach Previous rarity masses wash studies Cerithiidae Unidentified egg mass1 Rare F - 0 Superorder Caenogastropoda; Infraorder Littorinimorpha Planaxidae Hinea brasiliana Rare - Rare 0 Littorinidae Bembicium nanum Common F, A Common 6 Littorinidae Littorina acutispira Common - - 2 Littorinidae Littorina unifasciata Common - Common 6 Littorinidae Nodilittorina pyramidalis Common - Common 5 Rissoidae Rissoina sp. Uncommon - Uncommon 12 Rissoidae Rissoina (Rissolina)sp. Common - Uncommon 13 Vanikoridae Vanikora sigaretiformis Rare - - 0 Cypraeidae Cypraea annulus Rare - - 1 Cypraeidae Cypraea caputserpentis Rare - Rare 1 Cypraeidae Cypraea clandestina Rare - Rare 1 Cypraeidae Cypraea flaveola Rare - Rare 1 Cypraeidae Cypraea vitellus - - Rare 1 Cypraeidae Cypraea sp. Rare - - 14 Cypraeidae Cypraea juveniles Rare - Rare -6 (brown shell to 3-6cm)5 Cypraeidae Cypraea juveniles Rare - - 1 (white shell 1-2cm)7 Triviinae Trivia merces Rare - Rare 0 Cassidae Semicassis labiatum Rare - Uncommon 0 Ranellidae Cabestana spenglerii Common F Common 3 Ranellidae Charonia lampas Rare - - 2 Ranellidae Cymatium parthenopeum Rare - - 1 Ranellidae Ranella australasia Rare - Uncommon 0 Ranellidae Sassia parkinsonia Rare - Rare 0 Superorder Caenogastropoda; Infraorder Ptenoglossa Epitoniidae Epitonium sp. Rare - Rare 0 Epitoniidae Opalia australis Rare - Rare 0 Epitoniidae Opalia ballinensis Rare - Rare 0 Janthinidae Janthina exigua - - Rare 0 Janthinidae Janthina janthina - - Common 0 Superorder Caenogastropoda; Infraorder Neogastropoda Muricidae Agnewia tritoniformis Uncommon F, A Uncommon 2 Muricidae Bedeva hanleyi Uncommon F Rare 0 Muricidae Dicathais orbita Common F Common 5 Muricidae Ergalatax contracta Rare F - 0 Muricidae Lepsiella reticulata Uncommon F Uncommon 0 Muricidae Morula marginalba Common F Common 5 Muricidae Phyllocoma speciosa - - Rare 0 Muricopsinae Unidentified species - - Rare 0 Buccinidae Cominella eburnea Rare - Rare 0 Buccinidae Engina australis Rare - - 0 Muricidae Phyllocoma speciosa - - Rare 0 Muricopsinae Unidentified species - - Rare 0

1 This egg mass is too large to belong to Cacozeliana granaria and must therefore be from a different species of Cerithiidae. 2 Two species of Rissonia are recorded on the data base of shells from Shellharbour. These may not be the same species. 3 Refer to 2. 4 This is likely to be one of the large cowries recorded by Tarrant, 1980. 5 These are most likely Cypraea caputserpentis 6 Not sufficiently identified for comparison with previous studies. 7 These could be Cypraea vitellus (Tarrant, pers. comm.). There may be more than one species represented by these juveniles, some had a single yellow stripe across the shell. Chapter 2: Molluscan diversity 57

Family Species Regional Egg Beach Previous rarity masses wash studies Buccinidae Cominella eburnea Rare - Rare 0 Buccinidae Engina australis Rare - - 0 Buccinidae Fractolatirus normalis - - Rare 0 Buccinidae Nassarius jonasii Rare A Rare 1 Mitridae Mitra boudi Rare F Uncommon 0 Mitridae Mitra carbonaria Rare F Uncommon 0 Mitridae Mitra glabra - - Rare 0 Conidae Conus anemone Rare - Rare 0 Conidae Conus paperliferus Rare F Uncommon 0 Columbellidae Euplica versicolour Rare - Rare 0 Columbellidae Mitrella cf. semiconvexa Common - Common 0 Columbellidae Parviterebra sp. Rare - Rare 0 Marginellidae Austroginella sp. Rare - Uncommon 1 Marginellidae Volvarina mustelina Rare - - 0 Turridae Marita compta - - Rare 0 Unidentified Unidentified egg mass sp. 18 Common F - - Unidentified Unidentified egg mass sp. 29 Rare F Rare - Unidentified Unidentified egg mass sp. 310 Rare F - - Unidentified Unidentified egg mass sp. 411 Rare F - - Unidentified Unidentified egg mass sp. 512 - F Rare - Unidentified Unidentified egg mass sp. 613 - F Rare - Unidentified Unidentified egg mass sp. 714 - F Rare - Unidentified Unidentified egg mass sp. 815 - F Rare - Unidentified Unidentified egg mass sp. 916 - F Rare - Superorder Heterobranchia; Pulmonata Siphonariidae Siphonaria denticulata Common F, A Common 4 Siphonariidae Siphonaria funiculata Common - Common 1 Siphonariidae Siphonaria zelandica Common F, A Uncommon 0 Onchidiidae Onchidella patelloides Rare - - 0 Superorder Heterobranchia; Opisthobranchia Aplysiidae Aplysia dactylomela Rare F - 1 Aplysiidae Aplysia juliana Common F, A - 0 Aplysiidae Aplysia parvula Rare F, A - 0 Aplysiidae Aplysia sydneyensis Uncommon F, A - 0 Aplysiidae Dolabrifera dolabrifera Uncommon F, A - 0 Aplysiidae Stylocheilus longicauda Common F, A - 0 Aplysiidae Bursatella leachii Rare F - 0

8 Small lense shaped capsules primarily attached to Caulerpa filiformis. These could be the eggs of Mitrella sp. or Canthrindella picturata. 9 Hemispherical flat conjoined capsules. These are clearly prosobranch egg masses but the species responsible was not identified. It is possible they were laid by a species already listed for which the egg mass has not been identified. 10 Large aggregated mass of egg capsules. Possibly deposited by Semicassis labiatum (refer to Knudsen, 1992, for a description of a Cassidae egg mass). 11 Single white egg capsule with black egg. It is possible this was laid by a species already listed. 12 Tiny white compartmentalised egg capsules, found washed up on beach. It is possible these were laid by a species already listed. 13 Horny conjoined leathery capsules found washed up on beach. These are likely to be from a subtidal Muricid (refer to D’Asaro, 1991, for descriptions of Muricid egg masses). 14 Cup shaped egg capsules with frilly top and purple staining found washed up. These are likely to be from a subtidal Muricid (refer to D’Asaro, 1991, for descriptions of Muricid egg masses). 15 Flattened layer of conjoined egg capsules, found washed up on the beach. It is possible these were laid by a species already listed. 16 Balloon-like egg capsule with purple staining on a stalk, found washed up on the beach. These are likely to be from a subtidal Muricid (refer to D’Asaro, 1991, for descriptions of Muricid egg masses). Chapter 2: Molluscan diversity 58

Family Species Regional Egg Beach Previous rarity masses wash studies Philinidae Philine angasi Uncommon F, A - 0 Pleurobranchidae Berthellina citrina Rare F, A - 0 Pleurbranchidae Pleurobranchus peroni Rare F, A - 0 Pleurobranchidae Pleurobranchea sp. Rare F, A - 0 Bullinidae Bullina lineata Rare F, A Rare 0 Hydatinidae Hydatina physis Rare F, A Rare 0 Umbraculidae Umbraculum sp. Rare - - 0 Oxynoidae Oxynoe viridis Rare F - 0 Elysiidae Elysia australis Uncommon F - 0 Polybranchiidae Polybranchia orientalis Rare - - 0 Caliphyllidae Cyerce sp. Rare - - 0 Dendrodorididae Dendrodoris fumata Rare F, A - 0 Dendrodorididae Dendrodoris gemmacea Rare - - 0 Dendrodorididae Dendrodoris nigra Rare F - 0 Dendrodorididae Doriopsilla miniata Rare F - 0 Dendrodorididae Doriopsilla carneola Rare F, A - 0 Dorididae Discodoris fragilis Rare F, A - 0 Dorididae Platyodoris galbannus Rare F, A - 0 Dorididae Hoplodoris nodulosa Rare - - 0 Dorididae Jorunna pantherina Rare F, A - 0 Dorididae Jorunna sp. (purple) Rare - - 0 Dorididae Rostanga bassia Uncommon F - 0 Dorididae Glossodoris sp. Rare - - 0 Dorididae Thordisa sp. Rare - - 0 Chromodorididae Ceratosoma amoena Rare - - 0 Chromodorididae Hypselodoris bennetti Rare F - 0 Chromodorididae Mexichromis mariei Rare - - 0 Polyceridae Plocampherus imperialis Rare F, A - 0 Polyceridae Polycerid sp. Rare - - 0 Goniodorididae Goniodoris sp. Uncommon F - 0 Glaucidae Austraeolis ornata Rare F - 0 Glaucidae Glaucus atlanticus Rare - - 0 Glaucidae Glaucilla marginata Rare - - 0 Glaucidae Spurilla sp. Rare - - 0 Aeolidiidae Aeolidiella foulisi Uncommon F - 0 Unidentified Unidentified sp. 117 Rare F, A - 0 Unidentified Unidentified sp. 218 Rare - - 0 Unidentified Unidentified eggs sp. 1019 Rare F - 0 Unidentified Unidentified eggs sp. 1120 Rare F - 0 Unidentified Unidentified eggs sp. 1221 Rare F - 0 Unidentified Unidentified eggs sp. 1322 - F - 0 CLASS: POLYPLACOPHORA Ischnochitonidae Callochiton crocina Rare - - 0 Ischnochitonidae Ischnochiton australis Common - Uncommon 0 Ischnochitonidae Ischnochiton elongatus Common - - 1 Ischnochitonidae Ischnochiton versicolour Common - - 1 Chitonidae Sypharochiton pelliserpentis Common - Uncommon 4 Chitonidae Onithochiton quercinus Common - Uncommon 4

17 Small white species with green gills. 18 Black species with long tail and orange rhinophores. 19 Flattened white egg ribbon. These are clearly opisthobranch egg masses but the species responsible was not identified. It is possible that these were laid by one of the Opisthobranchs already listed. 20 Firm clear gelatinous ribbon with apricot eggs. 21 Large frilly yellow ribbon, possibly laid by several individuals. 22 Clear gelatinous rosette with yellow eggs. These eggs were collected subtidally and were not observed in the intertidal region. Chapter 2: Molluscan diversity 59

Family Species Regional Egg Beach Previous rarity masses wash studies Chitonidae Rhyssoplax jugosus Uncommon - Rare 0 Mopalidae Plaxiphora albida Uncommon - Uncommon 4 Acanthochitonidae Crytoplax mystica Rare - - 0 Acanthochitonidae Unidentified sp. 1 Rare - - 0 CLASS: BIVALVIA Mytilidae Mytilus edulis Rare - Rare 2 Mytilidae Brachidontes rostratus Rare - Rare 1 Mytilidae Xenostrobus pulex Common - - 0 Mytilidae Trichomya hirsuta Common - Uncommon 2 Pectinidae Scaeochlamys livida Rare - - 1 Anomiidae Anomia trigonopis Rare - Uncommon 0 Limidae Limatula strangei Rare - - 0 Spondylidae Spondylus cf. tenellus Rare - - 0 Montacutidae Kellia rotunda Rare - - 0 Ostreidae Saccostrea glomerata Common - - 0 Ostreidae Unidentified sp. 1 Rare - - 0 Unidentified bivalve Unidentified sp. 1 Rare - - 0 CLASS: CEPHALOPODA Octopodidae Hapalochlaena maculosa Rare - - 0 Octopodidae Octopus sp. Uncommon - - 0 Octopodidae Unidentified egg23 - F Rare 0 Idiosepiidae Idiosepius notoides Rare - - 0 Loliginidae Sepioteuthis australis - F Rare 0 Sepiidae Sepia sp. - - Uncommon 0 Total number of 17624 159 25 5926 9827 51 molluscs

Table 2.4: Species of mollusc found depositing egg masses in four natural estuarine habitats on the N.S.W. coast. SUPERORDER Species Site of egg Family deposition CAENOGASTROPODA Littorinidae Bembicium auratum Hard substratum Naticidae Polinices (Conuber) cf. sordidus Free in water column Muricidae Bedeva paivae Boulders HETEROBRANCHIA (PULMONATA) Basommatophora Salinator fragilis Shallow sand or mud Basommatophora Salinator solida Shallow sand or mud Unidentified Unidentified egg mass sp. 1428 Shallow mud

23 This egg capsules is clearly from an octopus but the species responsible was not identified. 24 161 of these are unambiguously distinct species. The juveniles of two species and the egg masses from 13 species were not sufficiently identified and could be represented by species already listed from adult specimens. 25 159 molluscs were recorded live in the intertidal area and 150 of these are unambiguously distinct species. The juveniles of two species and the egg masses of seven species are listed that could be already listed from adult specimens. The number of species recorded as:- Rare = 105; Uncommon = 21; Common = 33. 26 The egg masses from 58 species were found in the field and 24 species deposited egg masses in aquaria 27 17 species were only recorded from the presence of beach collected specimens. The shells of 10 species were recorded more commonly than live specimens and the shells of 10 species were less common than live individuals. 28 Muddy egg ribbons similar to Salinator spp. but thinner and deposited further upstream where no Salinator adults were found. Chapter 2: Molluscan diversity 60

egg masses were distinguished from molluscan egg masses by the absence of capsules surrounding the individual eggs.

2.4.2 Comparisons to previous studies Only 31% of the molluscs recorded in this study have been previously recorded on the Illawarra Coast (Table 2.3). A comparison between the number of molluscan species recorded during the inventories from this study and previous published surveys at the same site, revealed that a consistently greater number of molluscs were recorded in this study (Figure 2.5). The number of species recorded was highly under represented at three sites (Bellambi, North

Shellharbour and South Shellharbour), where I recorded a particularly high diversity of molluscs.

Most of the species that were not recorded in previous studies along the

Illawarra Coast were found to be regionally rare (78.8%, Table 2.5). Seventy five per cent of the common species were recorded in previous studies, compared to less than 18% of the rare species. A Chi squared test for heterogeneity revealed a strong significant difference in the likelihood that species were recorded in previous studies (χ2 = 36.06; p < 0.0001), based on the rarity recorded during this study in the Wollongong region. Nevertheless, eight species that were found to be common on the Wollongong coast during this study were not recorded in previous studies (Table 2.3, 2.5).

Chapter 2: Molluscan diversity 61

100

90 Inventory from this study Previous studies 80

Number of Species 30

0 South North North Bellambi Wombarra Scarborough Shellharbour Shellharbour Wollongong

Figure 2.5 : Comparison of the number of species of molluscs recorded in previous intertidal surveys in the Wollongong Region, with my records from a single survey at the same sites. The previous surveys at South Shellharbour and North Shellharbour were conducted by Marine Pollution Research, 1995; the survey at North Wollongong was done by Minchinton, 1996; the surveys at Bellambi was done by Miskiewicz and Lock, 1991; and the surveys at Wombarra and Scarborough were done by Marine Pollution Research, 1991.

Table 2.5: The number of regionally rare, uncommon and common species of mollusc that were recorded and had not been recorded in previous studies along the Illawarra Coast, N.S.W. Australia. The species include all clearly identified species that have been recorded live in the intertidal area along the Wollongong Coast during this study (refer to Table 2.3). Unidentified juveniles, egg masses and beach collected specimens were not included. The previous studies include:- Owen, 1978; Tarrant, 1980; Miskiewicz, and Lock, 1991; MPR, 1992; MPR, 1995; Minchinton, 1996; Australian Museum, 1997; Waters, 1997.

Regional rarity Recorded in previous studies Total number Yes No of species Rare 17 78 95 Uncommon 8 13 21 Common 24 8 32 Total no. species 49 99 148 Chapter 2: Molluscan diversity 62

2.4.3 Natural reefs vs. artificial habitats Most of the molluscs found live in the Wollongong region were recorded on natural intertidal reefs (98%; Figure 2.6). By comparison, only a small proportion

(31%) of species were recorded in artificial habitats (Figure 2.6). Three species

(Bursatella leachii, Philine angasi and Idiosepius notoides) were found in artificial habitats but not on natural reefs (Appendix 2.1, 2.2). The only

Opisthobranch molluscs found in artificial swimming pools were from the family

Alpysiidae, as well as, the estuarine species Philine angasi (Appendix 2.2). A slightly larger number of Opisthobranchs were found in the artificial channel compared to the pools (Appendix 2.2).

A high proportion of the egg masses recorded live (94%), were found on natural intertidal reefs (Figure 2.6). By comparison, only 29% of the egg masses were recorded in artificial habitats (Figure 2.6). More species were found breeding in the artificial channel at Wollongong than in the artificial swimming pools (Table

2.5; Appendix 2.3). These were all species that deposit egg masses on the underside of boulders (Table 2.5; Appendix 2.3). The egg masses from two species (Bursatella leachii and Philine angasi) were only recorded in artificial habitats and were not recorded on the adjacent reefs (Appendix 2.3).

2.4.4 Egg laying habitats Of the different substrata identified in the intertidal zone, boulders were the most commonly used for the deposition of molluscan egg masses. Nearly 60% of the molluscs found breeding on intertidal reefs along the Wollongong Coast deposit egg masses exclusively on the underside of boulders (Table 2.5). All of Chapter 2: Molluscan diversity 63

the boulders found with molluscan egg masses attached were at least partially submerged at low tide (pers. obs.). All of the egg masses that were deposited on the underside of boulders were found on sheltered, north facing reefs. By comparison, only 30% of the egg masses deposited under boulders were found on wave exposed reefs (Table 2.5). In general, the egg masses found under boulders on exposed reefs were in shallow pools or crevices high on the shore, where they were partially protected from strong wave action (pers. obs.).

Molluscs that deposit egg masses on exposed rock and/or algae were found on both sheltered and exposed reefs, as well as in artificial habitats (Table 2.5).

The species using exposed rock surfaces were primarily found in the high intertidal zone (Appendix 2.3). Dicathais orbita, differs from all other species by depositing egg capsules in the swash zone on vertical rock faces and rocky overhangs (Appendix 2.3). All of the Aplysiidae, except Dolabrifera dolabrifera, were found depositing egg masses on a range of substratum, including the walls of artificial pools (Appendix 2.3). Three species of Opisthobranch were found to embed their egg ribbons in sand (Table 2.5; Appendix 2.3). The egg masses from several subtidal molluscs were also found, including Cabestana spengleri, which broods egg masses in shallow subtidal caves (Appendix 2.3).

Two estuarine species were found attaching egg masses to hard substratum in natural estuarine environments (Table 2.4). Conuber cf. sordidus releases large egg masses into the water column and these are commonly found washed up on the shore. Three pulmonate molluscs deposit egg ribbons directly onto soft substrata (Table 2.4). Chapter 2: Molluscan diversity 64

180

160 Species 140 Egg masses

120

100

80 Total number 60

0 All intertidal reef habitats Natural Reefs Artificial habitats

Figure 2.6: The number of unambiguously distinct species of molluscs and molluscan egg masses recorded at thirteen natural reefs and nine artificial habitats on the Wollongong coast, NSW, Australia.

Table 2.5: Breeding sites used by intertidal reef molluscs along the Wollongong Coast, N.S.W. Australia. The number of species that attach benthic egg masses to the different types of intertidal substrata was recorded for naturally wave exposed and sheltered (north facing) reefs, as well as, artificial habitats, including swimming pools and Wollongong Channel. The species classified as using a particular substratum were exclusively found attaching egg masses to that surface. Whereas, the category multiple substrata includes species that utilise any combination of algae, boulders and exposed rock.

Substratum Number of species Exposed Sheltered Artificial Wollongong Total reefs reefs pools Channel Exposed rock 5 5 3 3 6 Under boulders 9 28 0 5 29 Algae 3 2 0 0 3 Multiple substrata 3 7 4 3 8 Sand 2 2 2 0 3 Total 22 44 9 11 49

Chapter 2: Molluscan diversity 65

2.4.5 Spatial and temporal variation in molluscan diversity The richness of molluscan fauna differs remarkably across different intertidal reefs on the Wollongong coast (Figure 2.7). Species lists for each site can be found in Appendix 2.1, with records of the species abundance and the occurrence of egg masses. The lowest diversity of molluscs was recorded at

Scarborough (29 species) and the maximum number of species recorded at one site was 131, at South Shellharbour (Figure 2.7). Generally, the species composition of molluscan taxa is similar across the sites (Figure 2.7). The greatest proportion of molluscs found, were prosobranch gastropods, at all sites. The number of Polyplacophora (chitons), bivalves, cephalopods and pulmonates did not vary greatly across the sites. However, the number of opisthobranchs and prosobranchs did vary.

The Opisthobranch molluscs, in particular, showed great temporal variation in their presence and abundance on intertidal reefs in the Wollongong Region.

This is reflected in the relatively large increase in the number of Opisthobranchs recorded in the cumulative surveys, compared to the numbers recorded in a single inventory at each intertidal site (Figure 2.7). Nine species of

Opisthobranch molluscs were only recorded on one occasion from a single reef

(Appendix 2.1). Eight of these species were recorded from a single individual and one species was recorded from just two individuals (Appendix 2.1). A further 12 species of Opisthobranchs were rare at every site they were observed, with less than ten individuals recorded along the Wollongong coast during the entire three year sampling period. The only common Opisthobranchs were a few species from the family Aplysiidae, which were found in large Chapter 2: Molluscan diversity 66

100 Cephalopods Bivalves 90 Chitons 80 Opisthobranchs 70 Pulmonates Prosobranchs 60

Number of Species 30

0 Be NS SS BP NW A Bu Cc Cd F S T W

Figure 2.7 a

Cephalopods 140 15 Bivalves Chitons 120 Opisthobranchs Pulmonates 100 19 3 Prosobranchs

80 2 14 20 19 60 9 15 10 16

Number of species 17 40 11

0 Be NS SS BP NW A Bu Cc Cd F S T W

Figure 2.7 b

Figure 2.7: The diversity of intertidal molluscs at a number of reefs in the Wollongong region from a) a single inventory of species at each site and b) cumulative surveys at each site. The number of surveys conducted at each site for the cumulative records is presented above the columns. The intertidal sites are:- Be = Bellambi, NS = North Shellharbour, SS = South Shellharbour, BP = Bass Point, NW = North Wollongong, A = Austinmer, Bu = Bulli, Cc = Coalcliff, Cd = Coledale, F = Flagstaff Hill, S = Scarborough, T = Towradgi, W = Wombarra. Chapter 2: Molluscan diversity 67

breeding colonies from late winter to early summer. All of the remaining

Opisthobranchs were uncommon and highly ephemeral, appearing in the intertidal zone in low numbers (3-10) for a few of months at the most and during this time they were often found with egg ribbons (data not presented).

Overall, the number of species recorded from each site increased with additional sampling periods (Figure 2.7, 2.9). However, the total species diversity at each site was not related to the number of surveys that were conducted (r = 0.110, p = 0.784; Figure 2.8a). There was a strong correlation between the number of species recorded in the single inventories and the total species diversity from the accumulated survey records (r = 0.969; p = 0.000;

Figure 2.8b). Nevertheless, the percent increase in species diversity from the inventory to the accumulated survey records was influenced by the number of surveys that were conducted (r = 0.567; p = 0.043, Figure 2.8c). In particular, there was a low percent increase in species diversity at North Shellharbour

(7.4%), which was only sampled twice, compared to all the other sites (average percent increase = 31.2%). The percent increase in species diversity at Bass

Point (29.0%), which was only sampled on three occasions, was not much lower than the average.

The cumulative species diversity has been plotted against the number of sampling events for each of the sites surveyed more than eight times (Figure

2.9). This indicates that the number of species tends to level off around 5 sampling events. However, at two sites, Shellharbour and Bellambi, new species were still being found after as many as 15 and 20 surveys respectively. Chapter 2: Molluscan diversity 68

See paper copy for Figure 2.8 graphs.

Figure 2.8: The relationship between sampling effort and the diversity of molluscs at 13 intertidal reefs on the Wollongong Coast NSW; a) the relationship between the total species diversity from cumulative surveys and the number of surveys that were conducted; b) the relationship between the number of species in a single inventory and the total species diversity from cumulative surveys; and c) the relationship between the number of surveys that were conducted and the percent increase in species diversity from the inventory to the accumulated species records. Chapter 2: Molluscan diversity 69

These two sites had the highest species diversity in both the inventory and the accumulated sampling records (Figure 2.7). A strong correlation was found between the species diversity and the percent of surveys that recorded additional species (r = 0.907; Figure 2.10a). However, the percent of surveys with new species does not appear to be influenced by the number of surveys that were conducted (r = 0.008; Figure 2.10b).

2.4.6 Molluscan diversity and the physical environment The diversity of molluscs, recorded during a single inventory of thirteen natural intertidal reefs in the Wollongong region, was compared to physical features of the reefs. These include exposure to wave action, the slope of the reef and habitat complexity. The rankings used are subjective and consequently, the results presented in this section should only be regarded as preliminary studies to direct future research.

Overall, a high correlation was found between the species diversity and the exposure to wave action (r = 0.868, p = 0.000). More species were found on sheltered reefs (ranked at five) than heavily wave exposed reefs (ranked at one; Figure 2.11a). Further support for the influence of wave exposure on molluscan diversity can be gained from a direct comparison of sheltered and exposed reefs with similar geographical features. The exposed reefs at

Towradgi and Bulli have a similar habitat complexity to the sheltered reefs at

Shellharbour and Bellambi (Table 2.1). A comparison of the species diversity at these sites reveals a much higher diversity at the two sheltered sites

(Figure 2.7). Chapter 2: Molluscan diversity 70

140

120

Be 100 SS NW A 80 Bu Cc Cd 60 F S

Number of species T W 40

0 1234567891011121314151617181920 Number of sampling events Figure 2.9: The cumulative species diversity of molluscs recorded on eleven intertidal reefs along the Wollongong Coast, NSW, Australia. The sampling sites are:- Be = Bellambi, SS = South Shellharbour, NW = North Wollongong, A = Austinmer, Bu = Bulli, Cc = Coalcliff, Cd = Coledale, F = Flagstaff Hill, S = Scarborough, T = Towradgi, W = Wombarra.

100 a 100 b 90 90

80 80

70 70

60 60

50 50

40 40

30 30

20 20

10 % surveys with additional species % surveys with additional species

0 0 0501001500 5 10 15 20 25 Total number of species Number of surveys Figure 2.10: The relationship between the percent of surveys in which new species of mollusc were recorded on an intertidal reef and; a) the cumulative species diversity; b) the number of surveys that were conducted. Chapter 2: Molluscan diversity 71

The diversity of molluscan fauna on intertidal reefs in the Wollongong region was also significantly correlated to habitat complexity (r = 0.784, p = 0.001).

Reefs with high habitat complexity (i.e. lots of boulders and a variety of microhabitats; ranked at five) supported a greater diversity of molluscs than rock platforms with few microhabitats (ranked at one, Figure 2.11c). Reefs with both high and low habitat complexity were found in wave exposed positions (refer to Table 2.2). In general, exposed reefs with high habitat complexity, such as Towradgi and Bulli, were found to have a greater diversity of molluscs than exposed reefs with low habitat complexity, such as

Scarborough and Austinmer (Figure 2.7).

Species diversity was also positively correlated to the slope of intertidal reefs

(r = 0.758; p = 0.003). A higher diversity of molluscs were found on gently sloping reefs (ranked at five) than on steep wave cut rock platforms (ranked at one, Figure 2.11b). However, slope appears to be confounded with habitat complexity (refer to Table 2.2). Consequently, habitat diversity could be influencing the observed correlation between slope and molluscan diversity. Chapter 2: Molluscan diversity 72

100

a 50

Number of species 30

0 0123456 Increasing shelter from wave action

100

70 b 60 50

Number of species 30

0 0123456 Increasingly gentle (horizontal) slope

100

70 c 60 50

Number of species 30

0 0123456 Increasing habitat complexity

Figure 2.11: The correlation between species diversity of intertidal molluscs and a) the degree of exposure to wave action (sheltered reefs are ranked at five and highly exposed reefs at one); b) the slope of the reef (gently sloping reefs are ranked at five and steep rock platforms at one); and c) the habitat complexity (highly complex reefs are ranked at five and rock platforms with minimal habitat complexity are ranked at one).

Chapter 2: Molluscan diversity 73

2.5 Discussion

2.5.1 Molluscan diversity and distribution in the Wollongong region The intertidal area along the Wollongong coast, N.S.W., Australia, was found to support a high diversity of molluscs. This study provides the most comprehensive record of the intertidal molluscs in the Illawarra Region, with over 100 species that have not been previously recorded (Table 2.3; Owen,

1978; Miskiewicz and Lock, 1991; Marine Pollution Research, 1992; 1995;

Minchinton, 1996; Waters, 1997). Nevertheless, the species diversity recorded in this study is likely to be considerably underestimated. Fifty species that were recorded in previous studies in the region have not been recorded during this study (Appendix 2.1). Most of these were listed in the

Australian Museum database from Shellharbour (Australian Museum, 1998). It has been estimated that there could be as many as 900 species of shelled molluscs represented in this museum collection (Ian Loch, per. comm.). Most of the museum specimens from Shellharbour were collected in the 1920s and a comprehensive database of this collection could be useful for examining historical changes in the distribution of Australian marine molluscs (refer to

Neilsen and West, 1994; Ponder, 1997).

This study has provided some new information on the distribution of marine molluscs in Australian waters. Most of the molluscs listed in Table 2.3 are endemic to Australia and there is a mix of tropical Indo Pacific and southern

Australian species (Wilson, 1993; Edgar, 1997). The majority of recorded species could be expected from the known distribution of marine molluscs in

New South Wales, Australia (refer to Iredale and McMichael, 1962; Bennett, Chapter 2: Molluscan diversity 74

1992; Wilson, 1993; Edgar, 1997). However, this study is the first N.S.W. record for at least one southern Australian nudibranch, Platydoris galbannus

(Willan, 1998) and two tropical prosobranchs (Tallorbis roseolus and

Astralium rhodostomum; Iredale and McMichael, 1962; Wilson 1993). One currently unnamed species, a Polycerid nudibranch (Bill Rudman, pers. comm.) was also found. The distribution of many other Opisthobranchs is not presently recorded.

It is apparent from this study that different intertidal reefs within a region are not equal in terms of molluscan diversity (Figure 2.7; 2.9). A particularly high diversity of molluscs was found on the north facing reefs at Shellharbour and

Bellambi. These two sites have been previously recommended as good sites for intertidal collecting (Pope and McDonald, 1961; McDonald, 1962).

Furthermore, Allan (1962) has suggested that Shellharbour is a hotspot of molluscan diversity on the N.S.W. coast. This contradicts the interpretation by

Marine Pollution Research (1995) of their findings in the Environmental

Impact Statement for the Shell Cove marina, which suggests that the coastal marine communities at Shellharbour are similar to other marine communities in the region.

2.5.2 Species rarity and implications for resource management Many of the intertidal molluscs recorded in this study were found to be rare in the Wollongong region (Table 2.3; 2.4). The proportion of rare species recorded in this study (64%) appears to be high compared to some previous studies on a range of different phyla (refer to Gaston, 1994, Table 1.4, pp 8- Chapter 2: Molluscan diversity 75

9). However, the cut-off points used to assign rarity are inevitably arbitrary and as a result, the proportion of species that are treated as rare can vary significantly between studies. For example, Gaston (1994) found that the proportion of species treated as rare in a particular assemblage ranged from

1.2 - 92.6%. In general, a higher proportion of rare species appears to be recorded in studies on macroinvertebrates (e.g. Goeden and Ricker, 1986;

Faith and Norris, 1989) than studies on vertebrates and plants (refer to

Gaston, 1994, Table 1.4).

It appears that rare species are unlikely to be detected in many intertidal surveys. Only a small proportion of the molluscs recorded as rare in this study, have been previously recorded along the Illawarra Coast (Table 2.5;

Appendix 2.2). Species rarity was found to significantly influence the likelihood that molluscs were included in previous species lists (p < 0.0001). However, the detection of rare species can be considered particularly important for the conservation and management of biological resources (Terborogh and Winter,

1990; Gaston, 1994, van Jaarsveld et al., 1998). The failure to detect rare species could lead to false assumptions about the value of the local environment (for example, refer to Section 2.5.1 and Marine Pollution

Research, 1995).

The interconnectedness of intertidal and subtidal regions could create problems for assessing the rarity of some intertidal species. Many of the molluscs recorded in this study may be more common in subtidal habitats. For example, Edgar (1997) describes the distribution of 38 molluscs recorded in Chapter 2: Molluscan diversity 76 this study, as extending into the subtidal region. Furthermore, several nudibranchs were only recorded on one-off occasions and these could be temporary visitors to the intertidal zone. From a conservation perspective, rare immigrants should usually be ignored because there are likely to be other areas that are more effective for their conservation. However, from an ecological perspective, immigrants should be regarded as a part of the community because they could contribute to a number of species interactions

(Gaston, 1994). It is also significant that several potentially subtidal

Opisthobranchs were only observed in small breeding aggregations in the intertidal area, during this study. Consequently, intertidal reefs could provide important egg laying habitat for some subtidal migrants.

It is generally though that a comprehensive system of marine reserves should provide for the special needs of rare species (e.g. Environment Australia,

1998). However, the location and design of reserves for the protection for rare species could be problematic, if these species do not occur in biodiverse

‘hotspots’ (Prendergast et al., 1993; van Jaarsveld et al., 1998). It is therefore significant that most of the rare molluscs found in this study were recorded on intertidal reefs that supported the highest diversity of species (refer to

Appendix 2.1). This is probably because rare species were a major contributing factor to molluscan diversity and therefore rare species are necessary for a site to be regarded as unusually species rich. Curnutt et al.

(1994) reported the coincidence of rare species and biodiversity hotspots for

Australian birds. However, these authors defined rarity at a coarse scale of spatial resolution (100 km2). On the other hand, rare species from a broad Chapter 2: Molluscan diversity 77 range of terrestrial taxa were not found to coincide with hotspots using spatial scales of 10 or 25 km2. The area of the intertidal reefs used in this study would all be considerably less than 1 km2 suggesting that a relationship between diversity and rarity could occur at a relatively small scale in intertidal ecosystems. Therefore the protection of highly diverse reefs could be an effective management strategy for regionally rare intertidal molluscs. Further studies on the relationships between rarity and species diversity would be of interest for marine conservation purposes.

It has been suggested that rare species should not be collected for bioprospecting purposes (Garson, 1996; International Union of Pure and

Applied Chemistry, 1996a; Workshop in Kuala Lumpur, 1996). Interestingly however, one study on molluscan natural products reports the collection of a rare species, Umbraculum mediterraneum (Cimino et al., 1988). Significantly, this large mollusc has a global distribution and only a single specimen was required for the characterisation of two biologically active compounds. This indicates that in some circumstances, chemical studies could be sensibly undertaken on rare species. However, this will depend on the sample requirements and the size of the species.

Rather than forbidding the collection of rare species it may be more appropriate to ensure that only a small proportion of the local population is collected. In this study, the size of the local population of molluscan species has been estimated, without quantitative sampling. Rapid assessment of species abundance is more likely to provide an underestimate, rather than an Chapter 2: Molluscan diversity 78 overestimate of the population size due to the fact that only a limited area will typically be surveyed. Therefore, rapid assessment should be useful for bioprospecting surveys.

The correct identification of species is essential for assessing species rarity and therefore the impacts of field collection for chemical studies. Of concern is a published study on an unidentified nudibranch, which reports the collection of 120g (12 pieces) of egg mass (Matsunaga et al., 1986). If these 12 ‘pieces’ represent 12 egg masses, it is possible that the entire next generation of the species was collected, at least on a local scale. Most of the nudibranchs recorded during this study were rare in the intertidal area on the Wollongong

Coast (Table 2.3), some with less than 10 individuals recorded over a three year period (Appendix 2.1). Since the actual species was not identified by

Matsunaga et al. (1986), it is impossible to assess the true impacts of their collection.

The egg masses of marine molluscs may be particularly vulnerable to overcollection. Only 10 common molluscs were found depositing egg masses on the intertidal reefs on the Wollongong Coast and a further four were found in estuarine environments (Table 2.3; 2.4). However, many species were found to deposit egg masses in aquaria, after being brought into the laboratory for positive identification (refer to Table 2.3). These include a number of rare Opisthobranchs, which appear to spawn in response to stress

(Rudman, pers. comm.). The adults of these species can be safely returned to the field. However, benthic egg masses are unlikely to survive after being Chapter 2: Molluscan diversity 79 detached from the artificial substrate. Consequently, molluscan egg masses deposited in aquaria can be used for chemical studies, without environmental impact above that caused by the collection of the species for accurate identification. Clearly however, eggs deposited in aquaria are lost to the natural ecosystem.

2.5.3 Molluscan breeding habitats Conservation reserves are particularly important for the protection of critical habitat, such as breeding grounds. In general, marine reserves are thought to act as centres for the dispersion of propagules and adults (refer to

Fairweather and McNeill, 1991; Allison et al., 1998). However, it can be difficult to demonstrate whether or not protected populations do act as a source of recruitment for the surrounding areas (Allison et al., 1998).

Consequently, the identification and protection of known breeding grounds should be a high priority for marine conservation.

Many intertidal molluscs in the Wollongong region were found to exclusively use sheltered sites for the deposition of benthic egg masses (Table 2.5).

Sheltered (north-facing) intertidal reefs are uncommon in the Illawarra area and therefore suitable breeding habitat for many molluscs in this region may be more restricted than generally thought (refer to Ponder 1998). Breeding aggregations in restricted areas make many marine organisms especially vulnerable to overexploitation and habitat impacts (Norse, 1993).

Consequently, these areas require special protection.

Chapter 2: Molluscan diversity 80

Parental site selection for the deposition of egg masses could be one factor influencing the distribution and abundance of molluscs on intertidal reefs. In this study, benthic egg masses from the greatest number of molluscs were found at the most species rich sites (i.e. Shellharbour and Bellambi; Appendix

2.1). These reefs provide a range of different intertidal substrata. However, most molluscs appear to attach their eggs to the underside of submerged boulders (Table 2.5). This may be a behavioural adaptation to sources of embryonic mortality such as desiccation, UV radiation and salinity stress

(refer to Fretter and Graham, 1962; Smith, 1972; Spight, 1977; Pechenik,

1979; 1986; Biermann et al., 1992). Consequently, the availability of boulders in suitable water-retaining hollows could be essential for the successful reproduction of many molluscs and therefore one factor affecting molluscan diversity in the Wollongong intertidal zone.

Parental site selection for the deposition of molluscan egg masses is likely to be the result of many factors influencing the evolutionary history of intertidal molluscs. Only a few molluscs adapted to live in the high intertidal area on wave exposed headlands, deposit benthic egg masses (Table 2.3; Smith et al., 1986). Most of these species attach their egg masses directly onto the rock surface (Table 2.5) and must therefore have evolved different mechanisms for reducing the effects of exposure to sunlight and air. Several molluscs deposit egg masses in rock pools or in the low intertidal zone on wave exposed reefs. These species use a range of substrata, including rocky overhangs and algae. Previous studies suggest that molluscan egg masses Chapter 2: Molluscan diversity 81 are less accessible to predators when deposited in these types of habitats

(Brenchley, 1982; Martel et al., 1986).

Overall, suitable breeding habitat for many intertidal molluscs appears to be limited on highly wave exposed coastlines. The egg masses from over half of the molluscs found breeding on the Wollongong Coast were only recorded on underside of boulders in sheltered (north-facing) intertidal reefs (Table 2.5).

This is probably because the suitability of boulders is influenced by wave exposure. Boulders on exposed reefs are subject to repetitive disturbance from wave action and burial by sand (McGuiness, 1984a; pers. obs.). The selection of boulders in sheltered areas could be an adaptation to minimise this type of disturbance, which could expose the eggs to unfavourable conditions.

2.5.4 Molluscan diversity and the physical environment It appears likely that the geographical features of an intertidal reef, influences species diversity. The greatest diversity of molluscs in the Wollongong region were found on gently sloping sheltered reefs, with a high degree of habitat complexity (refer to Table 2.2; Figure 2.7). These findings support the suggestions by Bennett (1992) and Allen (1975) that these types of reefs are the best sites for intertidal invertebrates. Intertidal molluscan diversity in the

Wollongong region was significantly correlated to shelter from wave exposure and habitat complexity. A higher diversity of species was found on sheltered reefs and more species were found on reefs with a greater diversity of microhabitats (Figure 2.11). A correlation was also observed between Chapter 2: Molluscan diversity 82 molluscan diversity and the slope of the reef. However, this factor could not be assessed independently from habitat complexity and therefore may not be a useful variable for comparing species diversity to the physical environment on intertidal reefs.

The relationships between molluscan diversity and the physical features of intertidal reefs must be interpreted cautiously in this study because the assessments were based on subjective rankings. Significantly however, Etter

(1989) found that qualitative assessments of wave exposure, based on distance from the headland, produced similar rankings to quantitative measures of maximum wave energies using a dynaometer. On the other hand, quantitative assessments of habitat complexity would be appropriate in future studies. The total area of each reef should also be quantified, as species diversity is commonly related to the area of available habitat

(McGuiness, 1984a). In particular, gently sloping reefs appear to have a greater area of intertidal habitat in comparison to steep rock platforms and therefore the greater diversity of these reefs could be due to their greater area.

Nevertheless, further evidence for the influence of geology on intertidal molluscan diversity is provided by the specific habitat requirements of many species. Edgar (1997) suggests that a large number of molluscs are restricted to sheltered or moderately exposed reefs. Most of these species were exclusively found on sheltered or moderately exposed natural reefs in this study (Appendix 2.1). On the other hand, all of the common molluscs that are Chapter 2: Molluscan diversity 83 adapted to live in wave exposed positions (e.g. Cellana tramserica, Turbo undulatus, Dicathais orbita; refer to Bennett, 1992; Edgar, 1997) were found on both sheltered and exposed reefs (Appendix 2.1). Consequently, sheltered intertidal reefs may support a greater diversity of molluscs because they satisfy a greater range of habitat requirements. This is consistent with the

Habitat Diversity Hypothesis (refer to Connor and McCoy,1979).

A high degree of habitat complexity is required to support a high diversity of molluscs, regardless of shelter from wave action. All of the naturally sheltered reefs in the Wollongong region have high habitat complexity. However, artificial swimming pools provide sheltered habitat with a low diversity of micro-habitats. Artificial habitats were found to support a much lower diversity of molluscan fauna than natural intertidal reefs (Figure 2.6). In particular, species that live or lay their eggs on the underside of boulders were not found in artificial swimming pools (refer to Table 2.5; Appendix 2.1; Edgar, 1997).

This provides further support for the applicability of the Habitat Diversity

Hypothesis to describing patterns of molluscan diversity on intertidal reefs.

Nevertheless, artificial habitats can increase the overall species richness on naturally exposed intertidal reefs by providing an additional type of habitat.

Sheltered areas of effectively subtidal sand can be found in artificial swimming pools. This type of habitat is more typical of an estuarine environment. In this study, three species were recorded in artificial habitats but not on natural reefs (Appendix 2.1). Two of these are estuarine species (Philine anagasi and

Idiosepius notoides) and the third (Bursatella leachii) prefers subtidal Chapter 2: Molluscan diversity 84 seagrass and sand habitats (Edgar, 1997). Consequently, artificial habitats can provide habitat for some estuarine and subtidal molluscs that do not normally occur on rocky intertidal reefs.

2.5.5 Methods for assessing intertidal molluscan diversity Species inventories, for the purpose of environmental impact assessment or resource management, are often performed by non-specialist ecologists using rapid biodiversity assessment. There are currently no guidelines for assessing the diversity of intertidal fauna in Australia. However, the diversity of species recorded in the intertidal area will vary according to the conditions of tide and swell, the survey methods, time spent searching for species and the expertise of the researcher. All of these variables could contribute to the large discrepancies in the number of species recorded during this study, compared to previous studies in the Wollongong region (refer to Figure 2.5). The standardisation of sampling methods is essential in rapid biodiversity assessment, for the cross-comparison of sites in time and space (Stork,

1994).

The use of existing information is a common approach to evaluating the significance of a research site (Lincoln Smith, 1991). For example, two previous studies on the Illawarra coast concluded that their study sites were similar to, or less diverse than other sites on the Illawarra coast, based on comparisons of their species lists with those from previous studies (MPR,

1995, pp vii; Waters, 1997, pp 16). However, both of these intertidal studies were conducted during high swell conditions and the authors did not compare Chapter 2: Molluscan diversity 85 the weather conditions or methodology used in previous studies. The diversity of molluscs recorded in one of these studies (MPR, 1995), was highly under- representative in comparison to the species inventory recorded at the same site in this study (Figure 2.5). Consequently, researchers should be cautious about drawing conclusions based on previous research and the limitations of the study should always be considered.

Most surveys for environmental impact assessment are conducted over a very short time period. These limited time frames provide only a static view of the environment (Fairweather, 1989; Lincoln Smith, 1991) and could potentially provide a misrepresentation of the total species richness in an area. However, the results from this study indicate that a high proportion of the intertidal molluscan diversity can be found during a single inventory of a site (Figure

2.7; Figure 2.9). Furthermore, the species composition of molluscan fauna in a single inventory appears be representative of the total composition after multiple surveys (Figure 2.7). There is a strong correlation between the species diversity in a single inventory and the cumulative diversity from multiple surveys (Figure 2.8b; r=0.97). This indicates that differences in molluscan diversity can be adequately assessed in a single comprehensive survey. No correlation was found between the overall species diversity and the number of surveys that were conducted (Figure 2.8a; r=0.11), although the percent increase in species diversity was influenced by the number of surveys (Figure 2.8c; r=0.53). Consequently, the actual number of surveys that are conducted is not important for the purpose of cross comparison but the number of surveys should be kept constant across sites. Chapter 2: Molluscan diversity 86

Although species inventories are useful for providing an estimate of the species diversity in an intertidal area, a comprehensive species list is much more difficult to obtain. This is because there is considerable temporal variability in species composition (Underwood, 1991; pers. obs.). In fact, new species were still being recorded after as many as 15 and 20 surveys at two highly diverse sites in the Wollongong region (Shellharbour and Bellambi;

Figure 2.9). The percentage of surveys in which additional species were recorded was significantly correlated to the overall diversity of molluscan fauna (Figure 2.10b; r=0.9), indicating that it is more difficult to obtain a comprehensive species list at highly diverse sites. Significantly, the species richness recorded in previous studies on the Wollongong coast was the most under-represented at highly diverse sites (Figure 2.5; Miskiewicz and Lock,

1991; MPR, 1995). Ultimately, the number of surveys that are required to obtain a comprehensive species list in the intertidal zone is not presently known. However, the number of surveys that are conducted will typically be imposed by restrictions in time and budget (refer to Warwick, 1993;

Fairweather, 1994).

Timing is likely to be a critical factor influencing the value of species inventories (refer to Lincoln Smith, 1991). Notably, the species inventories used in this study were all conducted on low spring tides with calm seas. This maximises the visibility and time available to search for intertidal organisms.

The comparatively high diversity of molluscs recorded in this study compared to previous studies at the same sites (refer to Figure 2.5), could be related to Chapter 2: Molluscan diversity 87 the use of optimal weather conditions. Species inventories in the intertidal area should always be planned ahead of time so that they are conducted during optimal conditions of tide and swell. Surveys should also be conducted at different sites in comparable tidal conditions to facilitate cross comparison.

The survey techniques employed could also influence the diversity of fauna found on intertidal reefs. All previous studies in the Illawarra region have used quadrats to count the diversity and abundance of species (Owen, 1978;

Miskiewicz, and Lock, 1991; MPR, 1992; MPR, 1995; Minchinton, 1996;

Waters, 1997). In this study a comprehensive survey of species richness was conducted by searching the entire range of microhabitats on the reef.

Abundances were not quantified but estimated for each site. This methodology is more likely to pick up rare species, such as the nudibranchs, which were generally found in low numbers in a localised region of the reef.

Less time is spent counting the number of individuals of a particular species and more time is spent searching for different species.

The use of qualitative rather than quantitative surveys has one disadvantage in that it is not possible to analyse differences in species abundance statistically. However, there is a high level of both spatial and temporal variability in the numbers of intertidal invertebrates (Underwood, 1991; 1992;

Underwood and Chapman, 1997) and consequently, a large number of replicate quadrats are required at each site, to obtain useful and meaningful comparisons. Furthermore, most environmental studies do not perform any statistical analysis on the data collected (Fairweather, 1994; Warnken and Chapter 2: Molluscan diversity 88

Buckley, 1998). The collection of quantified data on species abundance is clearly a waste of time, unless the data is to be used in the site analysis. In some circumstances this time might be better spent generating more comprehensive species lists. Quantitative data on species abundance should only be collected to test clear hypotheses that can be statistically analysed.

The range of phyla included in intertidal surveys could also influence the species diversity recorded within each phylum, by reducing the time spent on each group. The high diversity of intertidal flora and fauna, as well as the limited time available at low tide suggests that it may be appropriate to identify a group of biological indicators for intertidal studies. Stork (1994) provides a list of important criteria for selecting indicator organisms (Table 6.1, pp 91).

Marine molluscs appear to fulfil many of these criteria, but perhaps of greatest importance is the availability of taxonomic expertise compared to many other marine phyla. The Western Australian Museum and Conservation

International use marine molluscs as a key group in their rapid assessment surveys (Wells, 1998). These surveys are conducted by an expert in molluscan taxonomy. However, there are comprehensive illustrated reference guides for identifying shelled molluscs (e.g. Wilson, 1993) and recently the

World Wide Web has been used to facilitate the identification of

Opisthobranchs (Australian Museum Online Slug Forum, 1998).

The ease of identifying species by morphological differences is important for rapid biodiversity assessment. Previous studies using mosses and marine polychaetes have revealed that ambiguous morphological variation can lead Chapter 2: Molluscan diversity 89 to inaccurate biodiversity estimates (Oliver and Beattie, 1993; 1994). There are some species of cryptic marine molluscs that may cause underestimates of species diversity. For example, Wilson (1994) describes eight species from the genus Mitrella in N.S.W. waters, whereas I have only recorded one unidentified species of Mitrella in this study (Table 2.3). However, most macroscopic marine molluscs do have a distinct adult morphology (refer to

Beesley et al., 1998). Species with polymorphism and sexual dimorphism can also create problems for biodiversity assessment by producing an overestimate in the number of recorded species (Oliver and Beattie, 1993;

1994). Sexual dimorphism does occur in some marine bivalves (Morton et al.,

1998), although the phenomena appears to be generally uncommon in marine molluscs. Shell polymorphism is more common, although the variation within a species is likely to be illustrated and/or described in most texts used for molluscan identification (e.g. Wilson, 1993). Overall, marine molluscs are likely to produce fairly accurate estimates for rapid biodiversity assessment.

However, further research is required to determine whether molluscs are good surrogates for other intertidal phyla.

Beach debris can also be used to provide additional information about the distribution and abundance of shelled molluscs. The Western Australian

Museum includes the collection of empty shells in their surveys of molluscan diversity (Wells, 1998). In this study, seventeen species were only recorded from the presence of shells, indicating that these are entirely subtidal or open ocean species. Beach washed shells from a further 10 species were recorded more frequently that live individuals in the intertidal area (Table 2.3), Chapter 2: Molluscan diversity 90 suggesting these may be more common in subtidal habitats. However, beach debris is unlikely to be representative of the species diversity of subtidal molluscan fauna. For example, the shells of 10 intertidal species were found much less frequently in the beach debris than live individuals (Table 2.3) and these species all had fragile shells (pers. obs.). Only two species of soft- bodied mollusc were found washed up on the beach, which are both cephalopods that occupy offshore waters (Sepioteuthis australis and Sepia sp.; Bennett, 1992; Edgar, 1997). Consequently, the use of beach debris has little value for assessing the distribution and abundance of many molluscs, particularly Opisthobranchs.

Chapter 2: Molluscan diversity 91

2.5.6 Intertidal management recommendations for the Wollongong Coast Areas of high biological diversity (hotspots), such as the intertidal reefs on the northern side of Bass Point, Shellharbour, should be set aside in reserves.

The protection of sites containing a high diversity of species is considered essential for long term conservation in the marine environment (Fairweather and McNeill, 1991; Norse, 1993; Allison et al., 1998; Environment Australia,

1998). However, species diversity is probably best viewed as one of several criteria that should be used as a measure for conservation priority (Norse,

1993). It is therefore significant, that the intertidal reefs at Shellharbour also support many regionally rare molluscs and are used as a breeding ground by a range of species. Furthermore, all of the common intertidal molluscs recorded along the Wollongong Coast were found at Shellharbour (Table 2.2;

Appendix 2.1). This suggests that the molluscan fauna at this site is generally representative of that which occurs in the Wollongong intertidal area.

A high diversity of fauna could provide some indication of ecosystem health.

Consequently, reserves to protect biodiversity ‘hotspots’ may play a special role in regions subject to high human impact, such as intertidal reefs adjacent to urban areas (refer to Underwood, 1993; Keough, 1996; Dept. Environment

Sport and Territories, 1997). Allison et al. (1998) suggest that it may be important to isolate marine hotspots, as much as possible, from human threats. It is therefore significant that Bass Point is a coastal terrestrial reserve. The immediate protection of areas adjacent to existing reserves has been recommended in the International Convention on Biological Diversity,

1992 (article 8). Overall, the establishment of an intertidal protected area on Chapter 2: Molluscan diversity 92 the northern side of Bass Point appears to be a sensible management strategy for the Wollongong Coast, NSW, Australia.

2.6 Conclusion

A high diversity of intertidal molluscs was found along the Illawarra Coast,

N.S.W., Australia. In particular, a large number of rare species were found that have not been previously recorded in the region. A ‘hotspot’ of molluscan diversity was found at Bass Point, Shellharbour and this would be an appropriate site for the establishment of an intertidal protected area. The intertidal reefs at Shellharbour are sheltered from strong swell and have a high degree of habitat complexity. These physical features were positively correlated with high molluscan diversity and in particular, submerged boulders in protected areas are used for the deposition of benthic egg masses. Artificial habitats support a relatively low diversity of species, although these can increase the diversity of molluscs on wave exposed reefs by providing a subtidal or ‘estuary-like’ environment. Further research on the environmental factors affecting intertidal diversity could be used to rapidly identify hotspots of diversity along the Australian coast.

Species inventories can be used to rapidly assess the relative diversity of molluscan fauna at different reefs in a region. However, the diversity of molluscs recorded during intertidal inventories will depend on the survey techniques and the timing of surveys. Species inventories should be conducted in optimal weather conditions and quantitative sampling for species abundance should only be used to test clear hypotheses. Species abundance Chapter 2: Molluscan diversity 93 can be estimated to provide a rapid assessment of the likely environmental impacts associated with collecting species for bioprospecting purposes. In total, fourteen common intertidal molluscs were found depositing egg masses on the Illawarra Coast and these would all be suitable for chemical studies. A large number of other species were found to deposit egg masses in aquaria and these samples can also be used for preliminary antimicrobial screening.

NOTE

This online version of the thesis may have different page formatting and pagination from the paper copy held in the University of Wollongong Library.

UNIVERSITY OF WOLLONGONG

Chapter 3: Defence against predation and macrofouling 94

Chapter 3 Defence against predation and macro fouling on molluscan egg masses.

3.1 Introduction

The egg masses of marine molluscs are potentially susceptible to predation and overgrowth by macrofouling organisms. Molluscan egg masses can provide a considerable source of nutrients (Pechenik, 1979; Gallardo, 1979; Rivest, 1986;

Stöckmann-Bosbach and Althoff, 1989) and developing eggs are generally thought to represent an attractive source of food (Orians and Janzen, 1974).

Few molluscs provide parental protection for their eggs and benthic egg masses can be highly conspicuous (pers. obs.). The development of some encapsulated embryos can take many months (Laxton, 1969, West, 1973, Havenhand, 1993) and this long period of encapsulated development is likely to be sufficient for fouling from macro organisms to act as a significant selective pressure.

Consequently, many marine molluscs may have evolved mechanisms to reduce predation and surface fouling on their benthic egg masses. Nevertheless, the variability in resistance of egg masses to these selective pressures has never been specifically examined in marine molluscs.

3.1.1 Marine predators and predation on molluscan egg masses Reports of predation on molluscan egg masses are quite uncommon but there is some evidence to indicate that the egg capsules of prosobranch molluscs in general, are subject to predation (D’Asaro, 1970; Pechenik, 1979). Brenchley Chapter 3: Defence against predation and macrofouling 95

(1982) found that the egg capsules of the abundant estuarine snail Ilyanassa obsoleta were heavily preyed upon by crustaceans and inadvertently by an herbivorous gastropod. This predation was primarily perpetrated by an introduced species, while two native predators on molluscs were not found to consume the egg capsules of I. obsoleta. On the other hand Ilyanassa obsoleta was found to be an active predator upon the egg capsules of Cerithidea californica (Race, 1982).

The egg capsules from muricid gastropods are opened by a wide variety of predators, including opisthobranch and prosobranch gastropods, chitons, crabs, urchins, isopods, polychaetes and nemerteans (Haydock, 1964; Phillips, 1969,

Feare, 1970; Spight, 1977; Rawlings, 1990). Sea urchins have also been observed feeding on the egg masses of Neptunea antiqua and Buccinum undatum (Martel et al., 1986). A high proportion of the egg capsules deposited by Eupleura caudata were found to be damaged in the field and this was thought to be caused by predators (MacKenzie, 1961). Allan (1962) has noted that some molluscs will devour their own egg capsules and the eggs of other molluscs if they are starved in captivity.

There are fewer reports of predation upon the gelatinous egg masses of

Opisthobranch molluscs. Starfish were found to prey upon the egg masses of the sea hare Tethys californicus (MacGinitie, 1934). The egg ribbons of the nudibranch Hexabranchus sanguineus are known to be preyed upon by the

Aeolid nudibranch Favorinus japonicus (Scheuer, 1990). Aeolid nudibranchs have also been observed feeding on sea hare eggs in captivity (Allan, 1934; Chapter 3: Defence against predation and macrofouling 96

Rogers et al., 1995). Egg ribbons from the dorid nudibranch Glossodoris pallida were rapidly eaten by a number of fish in the field (Rodgers and Paul, 1991).

The gastropod Calliostoma annulatum has also been observed ingesting nudibranch egg masses (Perron, 1975). Humans are known to consume the egg ribbons from sea hares in Japan (Dr. Keiko Yamaguchi, pers. comm.). The sale of spawn from the sea hare Dolabella auricularia has also been observed in Fiji and the Society Islands (Rudman and Willan, 1998; Dr. Richard Willan, pers. comm.). However, the consumption of Aplysia egg masses is thought to cause diarrhoea and vomiting in humans (Miyamoto et al., 1988). The egg masses from three sea hares, Dolabella auricularia, Aplysia juliana and Aplysia kurodai, were universally rejected by crabs and reef fish (Pennings, 1994).

Cephalopod molluscs also deposit benthic egg masses that are potentially vulnerable to predation. Most octopus defend their eggs from predation

(Cousteau and Diole, 1973; Boletzky, 1986) and the Argonauta secrete a shell that provides additional physical protection for the eggs (Allan, 1939; Chung

Cheng and Dunning, 1998). However, cuttlefish and squid do not provide their eggs with parental protection. The eggs of cuttlefish and squid are considered to be inedible to fish, although the young cuttlefish are rapidly eaten as soon as they hatch (Cousteau and Diole, 1973).

3.1.2 Adaptations to predation: Behavioural and physiological defence. There are at least four ways in which the vulnerability to predation could be reduced in molluscan egg masses. These are by physical or morphological defence mechanisms, chemical defence, physiological defence and behavioural Chapter 3: Defence against predation and macrofouling 97

adaptations by the adult mollusc. Behavioural protection of the egg masses could involve brooding (e.g. Knudsen, 1950; Murray, 1964; Laxton, 1969;

Cousteau and Diole, 1973; D’Asaro, 1993) or parental site selection for the deposition of egg masses (e.g. Fretter and Graham, 1962; Boletzky, 1986;

Martel et al., 1986). Avoiding detection by potential predators could be the primary defence mechanism for many species (Karuso, 1987). This may involve unpredictable deposition of egg masses in space and time (refer to Bakus et al.,

1986) or cryptic spawning (e.g. Freame, 1940; Murray, 1964; D’Asaro, 1986;

1991). Physiological adaptations to reduce predation could involve low nutritional quality or rapid development. For example, some molluscan egg masses are known to hatch in as little as two days (Smith et al., 1986).

3.1.3 Physical defence against predation Neogastropods enclose their embryos within structurally complex proteinaceous capsules (Hunt, 1966; Bayne, 1968; Flower et al., 1969), which could provide physical protection from predation. There is great variability in capsule morphology (e.g. D’Asaro, 1991; 1993) and differences in these capsules could have implications for their protective value. For example, spines and overlapping ridges on the egg masses of some species of the Nassariinae, may form a protective barrier against predation (D’Asaro, 1993). The strength and thickness of the walls could also influence the effectiveness of egg capsules at preventing predation. The thick capsule walls of Nucella spp. were found to be more resistant to predation by isopods than thin capsules of the same species

(Rawlings, 1990; 1994). There is also some indirect evidence that predators of egg capsules have influenced the selection of strong but energetically Chapter 3: Defence against predation and macrofouling 98

expensive capsules walls in Conus spp. with long term embryonic development

(Perron, 1981; 1982).

Several species of prosobranch mollusc mould their egg capsules and assemble them into what has been described as “antipredatory masses”

(D’Asaro, 1970; D’Asaro, 1991; Knudsen, 1994). Thus some species may use a combination of physical and behavioural mechanisms to reduce predation on their egg masses. The compactness of the egg capsules in the egg mass of

Dicathais orbita is thought to deter crustacean predators that attack via the thin sides of the capsules (Phillips, 1969, D’Asaro, 1991). One species of

Nassariinae is also known to protect the thin side of capsules at the end of a row by attaching capsules that lack embryos (D’Asaro, 1993).

3.1.4 Chemical defence mechanisms The gelatinous egg masses of marine molluscs have no obvious physical protection from predation and it is likely that some of these are chemically defended. There is evidence that the conspicuous egg masses of sea hares contain chemical feeding deterrents. Extracts from the eggs of Stylocheilus longicauda deterred feeding by reef fish when incorporated into artificial foods

(Paul and Pennings, 1991). Extracts from the egg masses of Aplysia juliana were also found to deter feeding by reef fish in the field (Pennings, 1994). A lipid with laxative properties has been isolated from the egg masses of Aplysia kurodai (Miyamoto et al., 1988) and this compound could be responsible for the physiological reactions caused by the consumption of these eggs in humans.

Chapter 3: Defence against predation and macrofouling 99

The chemical defence of gelatinous egg masses against predation has only been investigated in two species of Opisthobranchs other than the Aplysiidae.

The egg ribbons of the ascoglossan Elysia halimedae contain very high concentrations of an alcohol that significantly deterred feeding by reef fish at natural concentrations (Paul and van Alstyne, 1988). Similarly, the egg masses of the nudibranch Hexabranchus sanguineus were found to contain a metabolite that is an effective feeding inhibitor against reef fish in the field (Pawlik et al.,

1988).

It has been suggested that there could be an inverse relationship between physical and chemical protection in molluscs (Faulkner, 1992a). However, some shelled molluscs do use a combination of physical and chemical defence, for example the limpet Collisella limatula (Pawlik et al., 1986) and the pulmonate

Trimusculus reticulatus (Rice, 1985). Consequently, it is possible that the physically protected egg capsules of neogastropods also contain chemical deterrents. Toxic chemicals have been found to reduce the palatability of egg masses from a wide range of invertebrates and effectively deter predation (e.g.

Euw et al., 1967; Lucas et al. 1979; Hare and Eisner, 1993; McClintock and

Vernon, 1990).

3.1.5 Predator feeding trials for assessing the role of chemical defence Chemical defence in marine invertebrates is often inferred from the ichthyotoxicity of their crude extracts or isolated metabolites. Many studies on marine molluscs use freshwater fish, such as goldfish or the mosquito fish

Gambusia affinis, to test for icthyotoxicity (e.g. Hellou et al., 1982; Thompson et Chapter 3: Defence against predation and macrofouling 100

al., 1982; Hochlowski et al., 1983; Ichiba and Higa, 1986; Cimino et al., 1988;

Bobzin and Faulkner, 1989; Miyamoto et al., 1995; Spinella et al., 1997). This type of study is not usually ecologically relevant and it is also difficult to relate toxicity to feeding deterrence. In fact, there is some evidence that toxic metabolites do not deter predators (Pawlik, 1993). A toxic metabolite would have little ecological advantage unless it does actually deter future attacks by the predator (Hay et al.,1987; Faulkner, 1992a). Schulte and Scheuer (1982), as well as, Faulkner (1992a) have noted that a number of studies on marine molluscs report the defensive value of isolated metabolites but do not present any quantitative data or experimental details on the antifeedant properties.

Field bioassays using natural predators are considered the most ecologically relevant experiments for testing chemical defence against predation (Faulkner,

1992a; Pawlik, 1993). However, these experiments typically require large quantities of the sample to be tested. As an alternative, aquarium bioassays with ecologically relevant predators can be used. However, these assays are dependant on the choice of predator (Faulkner, 1992a) and their generality may be limited. A number of experiments on molluscs have shown activity against some, but not all, potential predators (e.g. Rice, 1985; Pawlik et al., 1986;

Young et al., 1986; Rodgers and Paul, 1991). Ultimately, a combination of laboratory and field experiments would provide the most comprehensive assessment of antipredator defence. A combination of experiments has been previously used in several studies on marine molluscs (e.g. Pawlik et al., 1986;

Young et al., 1986; Rogers and Paul, 1991; Pennings, 1994; Rogers et al.,

1995). Chapter 3: Defence against predation and macrofouling 101

A range of methods have been used to test for feeding deterrent activity in marine molluscs. Whole organisms have been offered to predators both in the laboratory and the field (e.g. Rice, 1985; Pawlik et al., 1986; Young et al., 1986;

Manker and Faulkner, 1989b; Rodgers and Paul, 1991). Alternatively, extracts or metabolites isolated from molluscs have been offered to predators coated on pieces of squid mantle (Paul et al., 1990; Rodgers et al., 1995) or incorporated into an artificial feeding matrix (Rodgers and Paul, 1991; Pennings, 1994).

These artificial experiments permit an assessment of the palatability of a sample regardless of the physical structures or visible cues that could influence predator feeding. In most cases the natural concentration of extract is incorporated into artificial feeding disks by extracting a known volume of sample and mixing the extract into the same volume of food matrix (Pawlik, 1993).

However, the biological activity of a sample can vary according to the extraction procedure (de Vries and Hall, 1994). Becerro (1994) used an alternative artificial feeding experiment, which involved incorporating freeze-dried samples of sponge tissue into a feeding matrix. The chemical feeding deterrents are retained using this method (unless they are highly volatile) yet the physical structures are destroyed by grinding the sample into a fine powder.

Appropriate controls are essential for the analysis of feeding experiments. In artificial feeding experiments control feeding disks generally incorporate some form of dried food that make the feeding disks palatable. The same food is incorporated into treatment disks for consistency. Treatment and control feeding disks can then be offered to predators simultaneously so that the predators Chapter 3: Defence against predation and macrofouling 102

have the opportunity to express a dietary choice (i.e. preference experiments, see Peterson and Renaud, 1989). Alternatively, one group of predators could be offered controls and another group could be offered the treatments for a set period of time. This type of independent experiment enables a comparison of the feeding rate on different types of food, which can also reflect differences in the palatability of various foods (Peterson and Reaud, 1989). Preference experiments appear have been used more frequently, although Steinberg and van Altena (1992) have argued that this type of experimental design is not always the most appropriate. In both types of experiments controls for autogenic change are necessary (Peterson and Renaud, 1989; Roa, 1992). For example, the water content of the artificial matrix could change during the experiment and these changes could be different for control and treatments.

3.1.6 Macro fouling Every surface in the ocean is potentially a substratum for another micro- or macroscopic organism (Davis et al., 1989; Scheuer, 1990). In this chapter fouling is discussed exclusively with respect to macroscopic organisms. The role of microbial fouling on the surface of molluscan egg masses will be discussed in Chapter 4. Surfaces may be colonised by macroscopic fouling organisms in two ways (refer to Davis et al., 1989). Firstly, water borne adults, spores or larvae can settle on the surface. Alternatively, fouling could result from the lateral overgrowth of neighbouring organisms. Some fouling organisms are incapable of selecting suitable sites for settlement, for example the spores of red algae (Davis et al., 1989). On the other hand, the zoospores of green and brown algae, as well as the larvae of many invertebrates are capable of locating Chapter 3: Defence against predation and macrofouling 103

and settling on surfaces which are likely to improve their survivorship (Davis et al., 1989). The rapid development time and subsequent decomposition of many molluscan egg masses could make them unsuitable for colonisation by most fouling organisms.

A broad range of sessile marine organisms possess antifouling defences (refer to Bakus et al., 1986; Davis et al., 1989; Paul, 1992), suggesting that biofouling is an important selective pressure in the marine environment. An organism could be disadvantaged by epibiotic organisms if they compete with the host for food or increase the chances of the host organism being dislodged (Davis et al.,

1989). Dislodgment from the substrate could have detrimental effects on molluscan egg masses by increasing their susceptibility to sources of mortality associated with a planktonic existence (refer to Pechenik, 1979; Strathmann,

1985; Rumrill, 1990; Rawlings, 1994). However, competition for food is not likely to be a relevant selective pressure against fouling on molluscan egg masses.

This is because the nutritive reserves for the developing embryos are not on the surface of the egg mass but are enclosed within capsules inside the egg mass

(Pechenik, 1986; Gallardo, 1979; Stöckmann-Bosbach and Althoff, 1989).

Nevertheless, competition for oxygen could be a significant selective pressure on molluscan egg masses. Many encapsulated embryos are believed to be near the limit for adequate oxygen supply (Strathmann and Chaffee, 1984; Booth,

1995; Cohen and Strathmann, 1996). In both gelatinous egg masses and fluid filled egg capsules, ventilation depends on diffusion through all free surfaces

(Strathmann and Chaffee, 1984). Consequently, oxygen availability to Chapter 3: Defence against predation and macrofouling 104

molluscan embryos could depend on photosynthesis and respiration in the surrounding community. Any factor that restricts the availability or diffusibility of oxygen at the surface of egg masses could have a detrimental effect on the survival of molluscan embryos.

A green alga associated with salamander egg masses was found to have a profound effect on the supply of oxygen to the eggs (Bachmann et al., 1986). In light, oxygen was supplied to the amphibian eggs by photosynthesis, whereas respiration in the dark depleted oxygen availability. Similarly, unicellular algae and diatoms on the surface of polychaete and nudibranch egg masses were shown to effect the supply of oxygen to internal embryos (Cohen and

Strathmann, 1996). Even in shallow exposed positions favourable to photosynthesis, respiration by fouling organisms at night can result in hypoxia, which could retard or arrest the development of encapsulated embryos (refer to

Cohen and Strathmann, 1996).

Surface fouling could also have some potential benefits for molluscan egg masses. A coating of fouling organisms could effectively camouflage an organism (Davis et al., 1989) or reduce its palatability. Fouling could also protect embryos from solar radiation. Solar radiation on egg masses can be lethal to the embryos of some species (Biermann et al., 1992; Blaustein et al.,

1994; Rawlings, 1996).

Chapter 3: Defence against predation and macrofouling 105

3.2 Objectives

The purpose of this component of the study was to explore the mechanisms by which intertidal molluscs protect their egg masses from predation and macrofouling. There has been no previous assessment of the relative importance of chemical defence and other means of protecting molluscan egg masses, such as physical structures and behavioural adaptations. It is also not known how widespread the use of chemical feeding deterrents is in the egg masses of marine molluscs. The egg masses of seven species were tested for chemical feeding deterrents against selected predators in the laboratory and eight species were tested using predator feeding trials in the field (refer to Table

3.1). The species selected were all common in the intertidal area along the

Wollongong coast, with egg masses that were easy to collect in large quantities.

Independent and preference feeding experiments were used with predators in the laboratory but only preference experiments were run in the field due to the large quantities of material that would have been required. Observational studies on molluscan egg masses in the field were used to provide a preliminary assessment of any non-chemical defence mechanisms. Observations were also used to determine the significance of macrofouling as a selective pressure on molluscan egg masses.

3.3 Methods

3.3.1 Field and laboratory observations Observations on the egg masses of marine molluscs were made in the intertidal area along the Wollongong Coast, NSW (refer to Figure 2.1). The egg masses were examined for physical structures that could deter predation. For each Chapter 3: Defence against predation and macrofouling 106

species it was noted whether or not the eggs were clustered into a large aggregated mass. Aggregated egg masses were defined as an egg mass that was composed of multiple egg capsules or egg ribbons that were compacted into the smallest possible space. The individual egg capsules or egg ribbons, could have been deposited by one or several individuals, from the same species. Other breeding behaviours that were noted include parental protection for the developing embryos when the adult mollusc was found brooding the egg mass. Species that deposited cryptic or camouflaged egg masses were also noted. The egg masses were regarded as cryptic if they were difficult to see against the natural background and camouflaged if they were somehow concealed in the natural substrate. The egg masses of some species were collected and examined for signs of predation or fouling under a dissecting microscope (ANAX) at 4x – 25x magnification.

3.3.2 Collection and maintenance of the predators Crabs (Leptograpsis variegatus) were collected at night during low tide from the rock platforms around Flagstaff Hill, Wollongong (Chapter 2, Figure 2.1). These were bought back to the laboratory and placed in individual containers (1 Litre) in aerated seawater. The crabs were fed on bait prawns and left over night. In the morning the remaining prawns were removed, the crabs were placed in fresh sea water and starved for two days prior to the experiment. The water was changed every day before and during the experiment and the crabs were returned to Flagstaff Hill after the experiment was complete.

Chapter 3: Defence against predation and macrofouling 107

Isopods (Zuzara cf. venosa) were collected from the underside of boulders at

Towradgi or Wombarra reef (Chapter 2, Figure 2.1). These were bought back to the laboratory and placed in an aerated holding tank with pellets of fish food overnight. In the morning the water was changed and the isopods were starved for two days. During the experiment the isopods were individually placed in 5ml plastic tubes. The tubes were pierced 10 times around the bottom with a hot needle to allow water flow through the tubes. The tubes were floated on the surface of the water in aerated tanks by slotting them into pieces of polystyrene foam. This ensured that the isopods had the option of remaining fully submerged or unsubmerged. After the experiment the isopods were placed in the holding tank with clean water overnight and then returned to the site of collection.

Starfish (Pateriella calcar) were collected from Flagstaff Hill or Bellambi Reef

(Chapter 2, Figure 2.1). The starfish were placed in aerated individual containers (1L) and offered bait prawns and algae (Ulva lactuca), or agar feeding disks (refer to section 3.3.4) containing prawns, fish food or algae. None of the starfish appeared to consume any food in the individual containers, even after several days starvation. Therefore, the starfish were moved to a large (6ft) holding tank with aeration and filtration. The starfish were observed eating both prawns and algae in the large tank. The tank was split into two sections (control and treatment) and the starfish were starved for two days prior to the experiment. The starfish were returned to the site of collection at the end of the experiment. All predators were only used in one feeding trial before release. Chapter 3: Defence against predation and macrofouling 108

3.3.3 Collection and preparation of egg material The freshly laid egg masses of nine common intertidal molluscs were collected along the Wollongong Coast (Table 3.1). Notably, the egg ribbons of Salinator fragilis were laid during a red tide of dinoflagellates. The dinoflagellates coated the surface of these sticky eggs and it was not possible to remove them for the purpose of this experiment. All of the other egg masses were free from visible extraneous material.

Egg masses that were tested for antifeedant activity in artificial feeding experiments were freeze-dried and incorporated into an artificial feeding matrix

(refer to section 3.3.4). The frozen egg masses were ground in liquid nitrogen and placed in a freeze-drier until dry and brittle. The volume and weight of the eggs was calculated prior to freeze-drying. On average it took one day to freeze-dry 40ml of egg mass. The freeze dried egg masses were stored in a freezer (-200C) and then ground to a powder in a coffee grinder immediately before use. In a preliminary crab feeding experiment, the egg masses of A. juliana, B. nanum and D. orbita were also used fresh. The eggs were macerated in a Waring blender and then incorporated into an artificial feeding matrix without freeze-drying.

3.3.4 Preparation of artificial feeding disks. Feeding disks were prepared by incorporating bait prawns or dried fish food (K9 fish food for cold water fish, Friskies) into technical agar (Oxoid CM3). A set volume of the fresh egg mass was incorporated into an equal volume of agar.

Control disks consisted of the agar with prawns or fish food. Agar was dissolved Chapter 3: Defence against predation and macrofouling 109

Table 3.1: Molluscan egg masses collected for predator feeding trials. The

amount of egg mass collected and the site of collection are listed. The type of

feeding assay is recorded for each predator. Field feeding trials were conducted

in Wollongong Channel (WC) for reef species and Lake Illawarra (LI) for

estuarine species. The egg mass were either offered to the predators in their

natural state (N) or incorporated into an artificial feeding matrix and offered to

the predators in a preference (P) or independent (I) experiment.

Species Amount Site of Predators Feeding collected collection Assays Aplysia juliana 154.8g 200ml Bulli Pool Crabs N, P, I Isopods P, I Starfish N Field WC P Bembicium nanum 153g 180ml North Crabs N, P, I Wollongong Isopods P, I Field WC P Conuber c.f. sordidus 210.7g 180ml Lake Field LI P Illawarra Dicathais orbita 162.4g 200ml Towradgi Crabs N, P, I Isopods P, I Starfish N, I Field WC P Philine angasi 90.2g 150ml Bulli Pool Isopods P Field WC P Salinator fragilis 74g 50ml Lake Field LI P Illawarra Sepioteuthis australis 298.7g 300ml Towradgi Crabs P pool Isopods P Field WC P Siphonaria denticulata 10 egg ribbons Austinmer Crabs N Stylocheilus longicauda 78g 80ml Wollongong Isopods P Channel Field WC

Chapter 3: Defence against predation and macrofouling 110

in distilled water at 2.5% w/v and boiled in a microwave oven until clear. A concentration of 5% w/v was required in the field trial with the eggs of

Sepioteuthis australis to hold the disks together. The agar was cooled to 500C and prawns (20% w/v) or fish food (10% w/v) were added. The ingredients were thoroughly mixed and control disks poured. The egg material was then stirred into the remaining agar mixture when it reached approximately 400C. The treatment disks were poured and the agar was allowed to set.

3.3.5 Crab feeding experiments Seven crabs were starved for two days and then offered intact pieces of fresh, weighed egg mass from four species of mollusc (Table 3.1). Three crabs were offered a bait prawn as a control. The crabs were left overnight and in the morning the egg mass was reweighed. Three pieces of egg material from each species were placed in seawater overnight to calculate autogenic change.

Artificial feeding experiments were conducted with crabs using agar feeding disks in both independent and preference feeding experiments (Table 3.1).

Independent experiments involved twenty individually housed crabs that were randomly divided into two groups of 10 and starved for two days. One group was offered a control disk overnight and the other was offered a treatment disk.

Preference feeding experiments involved 10 crabs that were starved for two days and then simultaneously offered both a treatment and control disk overnight.

Chapter 3: Defence against predation and macrofouling 111

In preliminary independent crab feeding experiments fresh egg masses were incorporated into agar in 6ml petri dishes with prawns. The amount consumed was calculated by weighing the disks before and after being placed with the crabs. The weights were adjusted for autogenic change, which was calculated by placing both treatment and control disks (3 replicates) in seawater overnight without crabs.

In crab preference feeding experiments and one independent feeding trial with the eggs of Dicathais orbita, the freeze-dried egg material was incorporated into agar with fish food. The feeding disks were prepared by pouring the feeding matrix into plastic moulds (4cm x 2cm x 0.5cm) on strips of 1mm2 mosquito mesh (see Figure 3.1). These disks reduced the amount of egg material required for the feeding experiments and permitted a calculation of the amount consumed by area rather than weight. Autogenic control disks were left overnight in seawater without crabs.

3.3.6 Isopod feeding trials Artificial feeding experiments were conducted with isopods using the agar feeding disks in both independent and preference feeding experiments (Table

3.1). For independent feeding experiments, 20 individually housed isopods were randomly divided into two groups of 10 and starved for two days. One group was offered a control disk overnight and the other was offered a treatment disk.

For preference feeding experiments, 10 isopods were starved for two days and then each isopod was offered both a treatment and control disk overnight. In all Chapter 3: Defence against predation and macrofouling 112

experiments, four treatments and four control disks were placed in individual containers without isopods to calculate autogenic change.

Fish food was used in all feeding disks for the isopod feeding trials. Cylindrical feeding disks were prepared by punching holes with the wide end of a glass

Pasteur pipette from a 1cm deep agar mixture in a glass petri dish. These disks were weighed before and after the experiment to calculate the amount consumed. The weight of the disks was subsequently adjusted for autogenic change.

3.3.7 Starfish feeding trials In a large tank starved starfish were offered pieces of intact egg mass from

Dicathais orbita and Aplysia juliana by placing the starfish over the eggs. It was noted whether or not the stomachs of the starfish were everted on the egg material and the tanks were checked twice daily for regurgitated egg material.

An independent feeding experiment was set up using groups of ten starfish.

One group was offered one treatment disk each and the other group was offered one control disk each. The starfish were observed until each individual had consumed one disk. The tank was then checked for the next three days for regurgitated feeding disks. The starfish feeding disks were prepared in 6ml petri dishes with prawns.

Chapter 3: Defence against predation and macrofouling 113

3.3.8 Field feeding trials Field feeding trials were conducted in habitats in which the molluscs can usually be found. Field feeding trials using the egg masses of five intertidal reef molluscs were conducted in Wollongong Channel during calm sea conditions

(refer to Table 3.1). Field feeding trials were also conducted with the egg masses of two estuarine molluscs at Lake Illawarra (Table 3.1). One control and one treatment disk were attached to a single rope and the position was randomly determined. Twelve replicate ropes containing feeding disks were left in the field overnight. The ropes were anchored down with weights and a float was attached to the top of each rope (Figure 3.2). Initially lead weights were used to anchor the ropes but most of these were stolen during the first two experiments. Therefore the ropes were anchored using cement blocks in future experiments.

Four ropes containing autogenic control disks were also used in each experiment. The autogenic controls were enclosed in cages attached to the rope. The cages consisted of a cylinder of 2 x 2cm plastic mesh surrounded by

1x1mm mosquito mesh to prevent access by all macroscopic predators (Figure

3.2).

The feeding disks were prepared with fish food in 24 well polystyrene tissue culture plates (Corning Cellwells). The agar mixture was poured into the wells and allowed to partially set before the one end of a preweighed and labelled rubber band was dipped into each well. The agar was allowed to set around the rubber bands and then the disks were removed by gently by pulling them out with the band. The disks were weighed than attached to ropes using safety pins Chapter 3: Defence against predation and macrofouling 114

through the rubber bands. The amount consumed was calculated by weight and adjusted for autogenic change.

3.3.9 Statistical analysis Analysis of the independent feeding experiments were performed using t-tests.

A non-parametric Kruskal-Wallis t-test (Zar, 1984) was used when the data was not normally distributed and/or the variances were unequal. A Cochrans’ C test was performed to check if the variances were homogeneous. The preference feeding trials were analysed using two-tailed paired t-tests. A Wilcoxon paired- sample test was used for data that violated the assumptions of the parametric analysis (Zar, 1984). The amount consumed was calculated as a percentage of the initial weight or area of the feeding disk. Replicates where less than 5% of either disk had been consumed were excluded from the analysis. Replicates where both feeding disks were completely gone were also excluded from the field trials in case they had fallen off the ropes rather than been consumed. Chapter 3: Defence against predation and macrofouling 115

Figure 3.1: Artificial feeding disks containing fish food for a crab feeding trial using 1x1mm mesh to estimate the percent consumed based on area. A) control strip; B) treatment strip containing the freeze dried eggs of Aplysia juliana.

b c

Figure 3.2: Ropes containing artificial feeding disks (one control and one treatment randomly positioned) for a field feeding trial: a) lead weights; b) feeding disks; c) floats; and d) autogenic control cage.

Chapter 3 Defence against predation and macrofouling 116

3.4 Results

3.4.1 Defensive strategies against predation Intertidal molluscs were found to use a range of different strategies that could help reduce predation on their egg masses. Antifeeding activity was detected in the egg masses of five out of eight species tested for chemical defence in predator feeding trials (Table 3.2; refer to sections 3.4.2, 3.4.3, 3.4.4). The egg masses of 39% of the species appear to have some form of physical protection

(Table 3.2). The leathery egg capsules of the neogastropods (e.g. Figure 3.3;

3.4) was the most common type of physical defence. Most other species deposit soft gelatinous egg ribbons, which are often brightly coloured (e.g.

Figure 3.3; 3.5). The egg ribbons or egg capsules from 19 species were assembled into aggregated masses, which could function to reduce the effects of predation (Table 3.2; e.g. Figure 3.4; 3.5). The egg masses of five molluscs were cryptic or camouflaged (Table 3.2). These include the egg ribbons from two species of estuarine pulmonates (Salinator spp.), which were coated with mud or sand (e.g. Figure 3.6) and three species produce transparent egg masses (Nassarius jonasii, Philine angasi and Conuber c.f sordidus, Figure

3.5). Only one species (Cabestana spengleri), was found to provide parental protection for their developing embryos (Table 3.2) by externally brooding or guarding their egg masses.

Chapter 3 Defence against predation and macrofouling 117

Table 3.2: The proportion of molluscan species found using different strategies that may reduce predation on their egg masses along the Wollongong Coast, NSW, Australia. Chemical defence is determined from the results of predator feeding trials. All other features are determined by observations in the field. Parental protection could not be determined for the Cephalopods because these egg capsules were only found amongst beach debris.

Taxon Chemical Physical Aggregated Cryptic Parental defence defence egg mass spawn protection Neritopsina - 1/1 0/1 0/1 0/1 Littorinimorpha 1/2 1/4 2/4 1/4 1/4 Cerithiodea - 1/1 0/1 0/1 0/1 Neogastropoda 0/1 15/15 11/15 1/15 0/15 Pulmonata 1/1 0/4 0/4 2/4 0/4 Opisthobranchia 2/3 2/29 5/29 1/29 0/29 Cephalopoda 1/1 2/2 1/1 0/2 -

Chapter 3 Defence against predation and macrofouling 118

2cm

Figure 3.3: The aggregated egg masses of a) Agnewia tritoniformis and b) Mitra carbonaria under an intertidal boulder at Bass Point, NSW, Australia. The egg mass of Agnewia tritoniformis has been deposited by a number of different individuals and can be seen at several stages of egg development (i.e. yellow = freshly laid, brown = developing and purple = well developed). The egg mass of Mitra carbonaria is freshly laid and could have been deposited by one or several individuals. An adult Mitra carbonaria is also present in the bottem right hand corner.

2cm

Figure 3.4: The yellow gelatinous egg ribbons of Aplysia juliana are highly conspicuous on the walls of artificial swimming pools, such as Bulli Pool on the Wollongong Coast, NSW Australia. Several individuals from this species usually spawn communally to produce large aggregated masses.

Chapter 3 Defence against predation and macrofouling 119

2cm

Figure 3.5: The estuarine snail Salinator fragilis with it’s camouflaged egg ribbons (indicated by the arrow) at Towra Point Nature Reserve, NSW, Australia. The egg ribbon is embedded with mud as seen at 5x magnification (inset).

2cm

Figure 3.6: The transparent (cryptic) gelatinous egg mass of Conuber c.f. sordidus washed up on the shore at Lake Illawarra, NSW, Australia. Minute eggs are thinly dispersed through the large jelly mass.

Chapter 3 Defence against predation and macrofouling 120

3.4.2 Chemical defence: Crab feeding trials Crabs did not consume the intact egg masses of Dicathais orbita, Aplysia juliana, Bembicium nanum or Siphonaria denticulata when they were offered over night after two days of starvation. The starved crabs did however, consume the prawns overnight.

In the independent feeding experiments, crabs consumed the egg masses of

Dicathais orbita and Bembicium nanum but were deterred by the eggs of

Aplysia juliana, in an agar matrix containing prawns (Figure 3.7). Using a nonparametric t-test the crabs were found to consume significantly more of the control disks than disks containing the fresh egg masses of Aplysia juliana (p =

0.016; N=7). However, the probability values for the egg masses of Bembicium nanum (p = 0.652; N=9) or Dicathais orbita (p = 0.129; N=7) were not significant. Similarly, no significant difference was found between the amount consumed in control and treatment disks containing the freeze dried eggs of

Dicathais orbita with fish food, rather than prawns (p = 0.2669; N=6; Figure 3.7).

In the preference feeding trials using agar disks with fish food, the egg masses of three species Aplysia juliana, Bembicium nanum and Sepioteuthis australis showed strong antifeedant activity (Figure 3.8). However, the eggs of Dicathais orbita appeared to stimulate feeding by crabs (Figure 3.8). A Wilcoxon paired t- test confirmed that the crabs ate significantly more of the control disks than disks containing the eggs of A. juliana (p = 0.016; N=7), the eggs of Bembicium nanum (p = 0.016; N=7) and the eggs of Sepioteuthis australis (p = 0.023; N=

Chapter 3 Defence against predation and macrofouling 121

8). Conversely, significantly more of the treatment disks containing D. orbita egg masses were eaten than the control disks (p = 0.004; N=9).

Analysis of the amount consumed by area proved to be easier and less time consuming than analysis by weight. Significantly, there was no need to adjust the data for autogenic change using area. Autogenic control disks were placed in seawater overnight but there was no obvious change in the area of the disks.

In the first experiment where the percent consumed was calculated by weight there was an average increase of 0.05g in the autogenic controls. The average percent consumed was greater on the thin feeding strips measured by area than on the feeding disks measure by weight (Figure 3.7; Figure 3.8). This is because the thin feeding strips were of a smaller volume than the feeding disks that were prepared in petri dishes. This was also advantageous because a smaller amount of egg material was required for these experiments.

3.4.3 Chemical defence: Isopod feeding trials In the independent feeding experiments isopods were found to consume the feeding disks incorporating the freeze dried egg masses of Aplysia juliana and

Dicathais orbita but not the disks containing the eggs of Bembicium nanum

(Figure 3.9). A greater proportion of the control disks were consumed in all of these experiments (Figure 3.9). However, using a nonparametric t-test there was no significant difference in the amount consumed between control and treatment disks containing the eggs of Dicathais orbita (p = 0.352; N=8). The egg masses of Aplysia juliana were found to significantly deter feeding (p =

0.004; N=8) and the egg masses of Bembicium nanum strongly deterred feeding (p = 0.000; N=10).

Chapter 3 Defence against predation and macrofouling 122

90 Control 80 Treatment

70 N=6

50 N=7 N=9

40 ** N=7

percent consumed 30

0 Aplysia juliana Bembicium nanum Dicathais orbita Dicathais orbita Freeze dried Figure 3.7: Independent crab feeding trial using the fresh eggs of Aplysia juliana, Bembicium nanum and Dicathais orbita in an agar matrix with prawns. Controls consisted of prawns in agar only. The freeze-dried eggs of Dicathais orbita were also offered to crabs in an agar matrix containing fish food, with controls containing fish food only. The data are presented as the mean amount consumed with bars representing the standard error.

90 Control Treatment 80 ** ** ** ** N=7 N=7 N=9 N=8 70

Percent consumed 30

0 Aplysia juliana Bembicium nanum Dicathais orbita Sepioteuthis australis Figure 3.8: Preference crab feeding experiment using the freeze-dried egg masses from four marine molluscs in an agar matrix with fish food. Controls consisted of fish food only in an agar matrix. The data are presented as the mean amount consumed with standard error bars.

Chapter 3 Defence against predation and macrofouling 123

The egg masses from seven molluscs were tested for antifeeding properties against isopods in a preference feeding experiment. Overall, a greater proportion of the control disks were consumed than the treatment disks containing the eggs of all but two species, Dicathais orbita and Sepioteuthis australis (Figure 3.10). No significant difference was found in the percent consumed between control and treatment disk containing the egg masses of

Dicathais orbita (p = 0.407; N=10; paired t-test). The isopods appear to prefer feeding disks containing the eggs of Sepioteuthis australis over the controls, although the difference in the percent consumed was only significant at a 10% level (p = 0.08; N = 10; Wilcoxon paired t-test). Conversely, the egg masses of

Bembicium nanum were found to strongly deter feeding using a nonparametric paired t-test (p = 0.004; N=9). The egg masses Stylocheilus longicauda also significantly deter feeding by isopods (p = 0.039; N=9; Wilcoxon paired t-test).

Although, the egg masses of the other sea hare, Aplysia juliana did not significantly deter feeding in the preference experiment (p = 0.426; N=9;

Wilcoxon paired t-test). No significant difference was found in the percent consumed between control and treatment disk containing the egg masses of

Philine angasi (p = 0.154; N=10) or Conuber sordidus (p = 0.825; N=9) using a paired t-test. It should be noted that the control and treatment disks were difficult to distinguish using the egg masses of Philine angasi and Conuber sordidus. Consequently, the results for these species are somewhat subjective.

Furthermore, egg masses of Philine angasi were over six months old at the time of testing.

Chapter 3 Defence against predation and macrofouling 124

120

Control ** Treatment 100 N=8

N=8 80 ** N=10

40 Percent consumed

0 Aplysia juliana Bembicium nanum Dicathais orbita Figure 3.9: Independent isopod feeding trial using the freeze-dried eggs of Aplysia juliana, Bembicium nanum and Dicathais orbita in an agar matrix with fish food. Controls consisted of fish food only in agar. The data are presented as the mean amount consumed with bars representing the standard error.

80 Control Treatment 70 ** N=9

** * 40 N=9 N=10

N=9 30 N=10

percent consumed N=10

20 N=9

0 Aplysia juliana Bembicium Dicathais orbita Stylocheilus Philine angasi Conuber Sepioteuthis nanum longicauda sordidus australis Figure 3.10: Preference isopod feeding experiment using the freeze-dried egg masses from seven marine molluscs in an agar matrix with fish food. Controls consisted of fish food only in an agar matrix. The data are presented as the mean amount consumed with standard error bars.

Chapter 3 Defence against predation and macrofouling 125

In general, the autogenic control disks were found to decrease in weight by 1-

10% of the original mass. On the other hand most treatment disks increased in weight by 1-5%. In particular, the freeze-dried gelatinous egg masses of

Bembicium nanum and Aplysia juliana appear to absorb a lot of water. The amount of change in the weight of the autogenic disks varied by only 1-2% for replicate control and treatment disks during a single experiment. A greater variation was found in the amount of change between experiments and this depended on the length of time between removing the disks from the water and weighing the disks.

3.4.4 Chemical defence: Field feeding trials The egg masses of eight species of mollusc were tested for antifeedant activity in the field using a preference feeding experiment. Treatment disks containing the egg masses of five species were not consumed as readily as the control disks (Figure 3.11). Using a non-parametric Wilcoxon paired t-test, a significant difference was found in the amount consumed between control disks and treatment disks containing the eggs of Bembicium nanum (p = 0.031; N=6)

Aplysia juliana (p = 0.002; N=10) and Stylocheilus longicauda (p= 0.000; N=8).

There was a significant difference, at the 10% level, in the amount consumed using the eggs of Sepioteuthis australis (p = 0.082; N= 9). The egg ribbons of

Salinator fragilis also significantly deterred predation in an estuarine environment (p = 0.004; N=9). However, these eggs were coated in a dinoflagellate, which could have been responsible for the deterrent activity.

Proportionally more of the disks containing Dicathais orbita eggs were consumed than the controls (Figure 3.11). However, there was no significant

Chapter 3 Defence against predation and macrofouling 126

difference was found in the amount consumed using these eggs (p = 0.297;

N=7). The eggs of two species Philine angasi (p = 0.496; N=9) and Conuber c.f. sordidus (p = 0.416; N=10) were consumed in similar proportion to the control disks (Figure 3.11). However, the egg masses of Philine angasi were over six months old so the potential for feeding deterrents in these eggs should not be ruled out.

The autogenic controls in the field feeding trials generally increased in weight by a maximum of 5%. There was no more than a 2% difference in the autogenic change between the replicates in all experiments. The treatment and controls differed in the percent increase in weight by a maximum of 4%. Treatment disks containing the eggs of Bembicium nanum, Salinator fragilis, Stylocheilus longicauda and Sepioteuthis australis were found to absorb more water than the controls. The control disks for all of the other species showed a greater autogenic increase in weight.

3.4.5 Starfish feeding trials

In large aquaria Patiriella calcar were observed with their stomachs everted on the egg masses of both Aplysia juliana and Dicathais orbita. These eggs were drawn into the stomachs of a few starfish, although all the egg ribbons of

Aplysia juliana were regurgitated within two days. The egg capsules of

Dicathais orbita were also regurgitated but up to five days later. The egg capsules were regurgitated intact in all but one case where empty capsules were regurgitated.

Chapter 3 Defence against predation and macrofouling 127

50 Control

45 Treatment ** N=10 ** N=9 40

** N=6 35

N=7

30 N=9

** N=8

N=10 *

Percent consumed N=9 20

0 Aplysia juliana Bembicium nanum Dicathais orbita Stylocheilus longicauda Philine angasi Conuber sordidus Salinator fragilis Sepioteuthis australis

Figure 3.11: Field feeding trials with the freeze-dried egg masses of eight molluscs incorporated into an agar matrix containing fish food. The feeding disks were offered in a paired preference experiment using control disks containing fish food only in agar. The experiments were run in Wollongong Channel, except for the trials using the eggs of the estuarine species Conuber sordidus and Salinator fragilis. The estuarine feeding trials were run in Lake Illawarra. The data are presented as the mean amount consumed with standard error bars.

Chapter 3 Defence against predation and macrofouling 128

In the starfish feeding experiment with the eggs of Dicathais orbita in an artificial feeding matrix, both treatment and control disks were drawn into the stomach by the starved starfish. However, after four days all but one of the control disks and two of the treatment disks were regurgitated. No further feeding experiments were attempted with starfish.

3.4.6 Physical defence All neogastropods found along the Wollongong Coast, NSW, enclosed their embryos in tough leathery egg capsules (Table 3.2). The leathery egg capsules from several species were found washed up amongst beach debris with puncture holes (e.g. Figure 3.12), which were most likely to be created by predatory isopods. Puncture holes were always observed on the sides of the egg capsules and never on the top. Most neogastropods assemble a mass of egg capsules in to a tightly compacted space (Table 3.2; e.g. Figure 3.3), which would restrict access to the side of those egg capsules in the centre of the mass. The leathery capsules of some neogastropods have additional reinforcement, which could also deter some predators. The egg capsules of

Lepsiella reticularis have reticulated ridges on the top surface and the capsules of unidentified sp. 6 have spiky protrusions at the top. Unidentified sp. 5 produces egg capsules that are divided into several distinct compartments.

The egg masses from one Littorinimorpha (Cabestana spengleri) and one unidentified Cerithiodea have a leathery structure similar to the egg capsules of the neogastropods. However, some Littorinimorpha (e.g. Bembicium spp.) deposit gelatinous egg masses that appear more similar to the Heterobranchs

Chapter 3 Defence against predation and macrofouling 129

(Opisthobranchs and Pulmonates; Table 3.2). The egg ribbons of two species of

Opisthobranchs (Stylocheilus longicauda and Bursatella leachii) are tough relative to most other species in this group and these could deter predation by some small predators. The two Cephalopods also enclose their eggs in tough elastic structures (Table 3.2). The egg capsules of Nerita atramentosa are distinct from all other molluscan egg masses that were examined because they include calcareous structures, which could provide some physical reinforcement

(Figure 3.13). These small egg capsules are also firmly attached to the substrate making them difficult and time consuming to remove.

3.4.7 Macrofouling Macrofouling was only observed on the egg masses of eight out of 45 species of intertidal molluscs on the Wollongong Coast (Table 3.3). Most of these egg masses were fouled by macrophytes, in positions that were exposed to sunlight.

Green and blue-green algae were observed on the surface of both gelatinous egg ribbons and leathery capsules (Table 3.3). The only egg masses that were fouled on the underside of boulders were hatched leathery egg capsules (Table

3.3). Algal fouling was observed to be heavier on the hatched and well developed (purple) egg capsules of Dicathais orbita, than on the fresh (yellow) egg capsules of this species. Macrofouling invertebrates were only observed once, on the hatched leathery capsules of Conus paperliferus. These egg capsules were found on the underside of a boulder (Table 3.3).

Chapter 3 Defence against predation and macrofouling 130

Figure 3.12: The egg capsules of an unidentified prosobranch (sp. 7) at 10x magnification showing puncture holes (indicated by the arrows) that were most likely created by predatory isopods.

2cm

Figure 3.13: The egg capsules of Nerita atramentosa scattered across a boulder at Bellambi reef, NSW, Australia. The insert shows the calcareous structure of the egg capsules at 15x magnification.

Chapter 3 Defence against predation and macrofouling 131

Table 3.3: Macrofouling observed on the surface of molluscan egg masses along the Wollongong Coast, NSW, Australia. The type of egg mass and site of egg deposition is recorded for all egg masses that were fouled by macroscopic organisms. Hatched is used to indicate the macrofoulers were only observed on empty egg capsules.

Species Type of Site of Macrofoulers egg mass deposition Bedeva paivae Leathery On Encrusting blue-green algae capsules boulders Conus paperliferus Leathery Under Sponge (hatched) capsules boulders Encrusting bryozoan Spirorbid worm tubes Dicathais orbita Leathery Vertical Filamentous green algae capsules rock walls Encrusting blue-green algae Branched brown algae Encrusting red algae Filamentous red algae Unidentified prosobranch Leathery Under Encrusting blue-green algae (sp. 2; hatched) capsules boulders Encrusting red algae Salinator fragilis Gelatinous Muddy Dinoflagellates ribbons surfaces Siphonaria denticulata Gelatinous Rock Green algae ribbons platforms Encrusting blue-green algae Aplysia juliana Gelatinous Pool walls Filamentous green algae ribbons Encrusting blue-green algae Aplysia sydneyensis Gelatinous Pool walls Filamentous green algae ribbons

Chapter 3 Defence against predation and macrofouling 132

3.5 Discussion

3.5.1 Defensive strategies against predation The intertidal molluscs on the Illawarra Coast, NSW, Australia, employ a variety of strategies that could help reduce the impact of predation on their egg masses. Only one species (Cabestana spengleri) was found to provide parental protection for their eggs by brooding the egg mass (Table 3.2). This species has a relatively long period of encapsulated development (Laxton,

1969). All the Neogastropods and some species from other taxa enclose eggs in leathery egg capsules, which potentially provide physical protection for the developing embryos (Table 3.2). The egg masses of a number of other species showed no sign of physical protection and some of these appear to be chemically defended. Other parameters identified that may reduce predation on the eggs of intertidal molluscs include camouflage and the deposition of large aggregated egg masses (Table 3.2).

Leathery egg capsules may negate the requirement for chemical antifeedants in Neogastropod egg masses. The egg mass of Dicathais orbita was not found to deter feeding by intertidal predators, after the physical structure of the egg capsules had been destroyed. The capsule walls of gastropod egg masses are thought to be of low nutritional quality and indigestible to predatory crabs, gastropods and isopods (Brenchley, 1982; Rawlings, 1994).

Nevertheless, leathery egg capsules do appear to be susceptible to predation by some small intertidal predators, such as isopods (Figure 3.11; Rawlings,

1990; 1994). This may explain why many Neogastropods assemble their egg capsules into tightly compacted aggregated masses (Table 3.2; e.g. Figure

Chapter 3 Defence against predation and macrofouling 133

3.3). Aggregated egg masses could function to reduce predation by providing safety in numbers and by limiting predator access to vulnerable parts of the egg capsules (refer to Phillips, 1969, D’Asaro, 1991).

The gelatinous egg masses of the Heterobranchia and some Littorinimorpha have no obvious form of physical protection and some of these appear to be chemically defended against intertidal predators (Table 3.2). I found evidence of chemical feeding deterrants in the egg masses of two common intertidal molluscs (Aplysia juliana and Bembicium nanum; Figure 3.8, 3.9, 3.10, 3.11).

Antifeeding activity has been previously reported in the extracts from

Aplysiidae egg masses (Paul and Pennings, 1991; Pennings, 1994) and chemical feeding deterrents have been isolated from the egg mass two

Opisthobranchs (Paul and van Alstyne, 1988; Pawlik et al., 1988).

Consequently, some molluscs appear to use chemical feeding deterrents as the primary defence mechanism against predation on their egg masses.

Nevertheless, a combination of chemical and physical feeding deterrents may be used by some species (Table 3.2). Both Stylocheilus longicauda and

Sepioteuthis australis enclose their eggs in tough elastic structures, which could deter small predators. Nevertheless, these egg masses were found to deter isopods and intertidal predators in the field, after the physical structure had been destroyed (Figure 3.10; 3.11).

On the other hand, there was no evidence of physical or chemical defence in the egg mass of the Littorinimorpha Conuber c.f. sordidus or the

Chapter 3 Defence against predation and macrofouling 134

Opisthobranch Philine angasi (Table 3.2; Figure 3.10; 3.11). The transparent appearance of these egg masses may provide some protection by making them difficult to detect in the water column (e.g. Figure 3.6). It is also possible that these egg masses are of low nutritional quality. The eggs of these species are small and thinly dispersed in a large mass of jelly (pers. obs.).

Previous studies have shown that egg mass jelly is generally of little nutritional value (Pechenik, 1979). In fact, the egg mass of Conuber sordidus has been found to consist of as much as 93% water (Murray, 1964). Conuber sordidus embryos are also know to hatch in as little as three days (Smith et al., 1986), which means that there is a high probability that some embryos from each egg mass will escape predation. Chemical or physical defence of egg masses may not be required for some species with rapid embryonic development.

Some molluscs may provide further protection for their eggs by incorporating extraneous material into their egg masses. Egg capsules with calcareous inclusion bodies appear to be typical for species of the genus Nerita (Figure

3.13, Knudsen, 1997c). The inclusion bodies, which could be derived from shell fragments or other calcareous skeletons, appear to reinforce the capsule wall of these egg masses. There is some evidence that calcareous intrusions in gorgonians and soft corals dissuade predators (Harvell et al., 1988; van

Alstyne and Paul, 1992; van Alstyne et al., 1992; 1994). On the other hand, the estuarine pulmonates (Salinator spp.) used sand and mud to coat their egg masses. This could provide an effective camouflage for the eggs (Figure

3.5), in addition to deterring predators. Wright et al., (1997) have suggested

Chapter 3 Defence against predation and macrofouling 135 that the incorporation of sand into sponges could make them unpalatable to predators. The incorporation of inorganic material into molluscan egg masses could act directly as a feeding deterrent or indirectly by reducing the nutritional quality of the egg mass.

The apparent rejection of feeding disks containing the egg ribbons of Salinator fragilis, by estuarine predators in the field, could also be due to the presence of dinoflagellates on the surface of the eggs. Dinoflagellates, are known to be toxic to fish and other marine life (LoCicero, 1975). It is of interest that this population of Salinator fragilis was only found spawning during the time of the toxic bloom but not in the few weeks prior to following the bloom. Therefore, it remains possible that this population was capitalising on the toxicity of the dinoflagellates to provide further protection for their eggs.

3.5.2 Chemical defence and artificial feeding trials In this study artificial feeding experiments were used to examine the relative palatability of molluscan egg masses. In general, it was found that these experiments were useful for assessing the importance of chemical defence against predation. However, feeding experiments typically require large amounts of sample material, which essentially restricts their applicability to common species. It is reasonable to assume that common molluscs may, in part, owe their success to the ability to overcome the impacts of predation on their egg masses. However, common species may not be representative of any of the less common molluscs because a different set of selective pressures could be operating for these species. Consequently, whilst predator

Chapter 3 Defence against predation and macrofouling 136 feeding trials are useful for assessing the evolutionary success of particular common species, they can not be extrapolated to produce general conclusions about the relative importance of chemical feeding deterrents in the different types of molluscan egg masses.

There are also a number of problems associated with conducting artificial feeding experiments. First, the samples are tested for palatability in an artificial feeding matrix, which may not reflect the true nutritional quality of the original sample. Experiments on the metabolites from sponges and algae have shown that chemical defences are less effective in high quality than low quality foods (Duffy and Paul, 1992; Pennings et al., 1994). In my experiments, the whole egg masses were incorporated into an artificial feeding matrix with a second source of nutrients, for example fish food. This was necessary to ensure that the controls were palatable and to provide consistency between the controls and treatments. However, the combination of both eggs and fish food into the treatment disks means that the nutritional quality of these disks is higher than naturally occurs in the egg masses.

Furthermore, nutritional quality would be higher in the treatment disks with the eggs, than the controls, which only contained fish food. Consequently, these feeding experiments only provide a conservative test for the palatability of molluscan eggs and a lack of observed activity does not rule out the potential for chemical defence. In particular, the egg masses of Conuber sordidum and

Philine angasi did not deter feeding by isopods or in the field, when incorporated into artificial matrices (Figure 3.10; 3.11) and these egg masses are likely to be of lower nutritional quality than the artificial feeding disks.

Chapter 3 Defence against predation and macrofouling 137

Ultimately, the nutritional quality of an artificial feeding matrix should be controlled to reflect the nutrient availability in the natural sample.

The higher nutritional quality of the treatment disks could explain why the egg masses of Dicathais orbita were consumed at a significantly higher rate than the control disks, when offered to crabs in a preference experiment (Figure

3.8). Differences in nutritional quality may also explain the different results that were obtained in two crab feeding experiments with the eggs of

Bembicium nanum (Figure 3.7; 3.8). In the first experiment fresh eggs were used with prawns, whereas in the second experiment freeze-dried eggs were used with fish food. The overall concentration of Bembicium eggs would have been much higher in the second experiment where significant feeding deterrence was found. Furthermore, it is possible that prawns mask the antifeedant properties of the Bembicium eggs in the first experiment due to the high nutritional qualities or a stronger flavour. Alternatively, the different results in these experiments could be due to a different experimental design

(see below).

Chemical feeding deterrents may not be equally effective against all marine predators. The detection of chemical feeding deterrents can depend on the choice of predators (Faulkner, 1992a). In this study it was found the egg masses of Aplysia juliana and Sepioteuthis australis significantly deter feeding by the intertidal crab Leptograpsus variegatus and most natural predators in the field (Figure 3.7; 3.11). However, these egg masses appear to be palatable to isopods (Zuzara sp.) in an artificial feeding matrix (Figure 3.10).

Chapter 3 Defence against predation and macrofouling 138

Isopods can be found in relatively high densities in the intertidal region (pers. obs.). However, they are small and therefore unlikely to have a significant impact on the reproduction rate of many large and prolific molluscs, such as

Aplysia juliana. In general, it has been assumed that field feeding trials are the most ecologically relevant experiments for testing chemical defence against predation (Faulkner, 1992a; Pawlik, 1993). However, there is great temporal and spatial variation in the biological assemblages in the marine environment

(Underwood, 1991; 1992; Underwood and Chapman, 1997). Consequently, a single feeding experiment may not always reflect the natural patterns of predation. Laboratory feeding experiments can therefore provide additional information about the potential effects of selected ecologically relevant predators.

One further question with predator feeding trials concerns the relative merits of conducting independent (separate choice) or preference experiments. In this study the relative consumption of some egg masses did vary using the same predator in the different types of experiments. This is consistent with the findings of Steinberg and van Altena (1992) in experiments using marine herbivores. It has been argued that the interpretation of the results from independent feeding experiments can be misleading because no choice is actually offered (Roa, 1992). Preference experiments appear to have an advantage over separate choice feeding experiments because they allow a direct comparison of the rate of consumption between a choice of foods.

However, independent feeding experiments would be more appropriate in cases where the predator does not get the opportunity to make simultaneous

Chapter 3 Defence against predation and macrofouling 139 choices between prey. This scenario may be realistic in circumstances where prey is patchily distributed over a large area and the predator has limited motility.

Independent feeding experiments provide information on the feeding behaviour of potential predators when there is no other food available. In this study, feeding disks containing the egg mass of Bembicium nanum were consumed by crabs when no choice was offered but they significantly deterred feeding by crabs in a preference experiment (Figure 3.7; 3.8).

Notwithstanding the problem of altered nutritional quality (see above), these results indicate that the eggs of Bembicium nanum can be consumed by starving crabs, although they prefer not to if other food is available. By comparison, only a small proportion of feeding disks containing the egg ribbons of Aplysia juliana were consumed by crabs in both independent and preference feeding experiments (Figure 3.7; 3.8). This suggests that the chemical deterrents in the egg mass of Aplysia juliana are strong enough to deter feeding by crabs under all circumstances. On the other hand, the eggs of Aplysia juliana were consumed at a significantly lower rate than the controls in an independent isopod feeding experiment but not in a preference experiment (Figure 3.9; 3.10). This result may be due to chance, as small sample sizes were used in the experiments. Alternatively, it could reflect population variation in the chemical properties of Aplysia eggs.

Controlling for autogenic change is one problem with predator feeding trials that has received some attention in the literature (Peterson and Renaud,

Chapter 3 Defence against predation and macrofouling 140

1989; Roa, 1992). In general, low variation between the autogenic controls was found during this study and the overall changes were fairly insignificant

(i.e. 1-5% of the initial weight). The method that I have used to adjust for autogenic change has previously been criticised in the literature (Peterson and Renaud, 1989; Roa, 1992). However, the methods that have been recommended by these authors require an equal number of autogenic controls and treatment replicates. When working with biologically valuable material, such as molluscan egg masses the collection of any amount of sample has to be justified. In this study, I decided the collection of large amounts of egg material to facilitate greater replication of autogenic controls was unjustified. Overall, the amount of autogenic variation observed in this study was small and is therefore unlikely to affect the results, especially where strong significant differences were found.

3.5.3 Potential physical defence against predation The effectiveness of physical protection afforded by the leathery capsules of marine molluscs could vary according to the structure of the capsule wall and the available predators. Leathery egg capsules may significantly reduce predation, although they are unlikely to be completely effective against all intertidal predators. For example, isopods have been found to chew through the capsule walls of Nucella spp. and Rawlings (1990; 1994) found that the thin walled capsules of Nucella spp. were more readily consumed by isopods than thick walled capsules of the same species. Thin walled capsules were only found at sites without isopods (Rawlings, 1990), suggesting that isopods could have influenced the evolution of thick walled capsules. Further studies

Chapter 3 Defence against predation and macrofouling 141 on the relative thickness and ‘toughness’ of molluscan egg capsules would shed some more light on the role of physical defence in the protection of molluscan embryos.

The leathery egg capsules of several prosobranchs molluscs on the Illawarra

Coast were found to have additional morphological features, which could improve their effectiveness against intertidal predators. These include the spiky projections and reticulated ridges on the top surface of two

Neogastropod egg capsules. Similar morphological structures have been previously described and implicated in the protection of gastropod eggs against predation (D’Asaro, 1993). However, there appears to be no previous reports of the internal segregation of the molluscan egg capsules into distinct compartments, as was found with the egg capsules of one unidentified prosobranch (sp. 5). Segregating the egg capsules into distinct compartments could greatly improve the chance that some embryos will survive an attack from small predators, such as isopods. However, this type of reproductive structure could be rare because it is energetically expensive to produce. In most cases, the evolution of physically protective reproductive structures probably results from a balance in resource allocation between the eggs and the egg capsule (refer to Perron, 1982).

3.5.4 Macrofouling on molluscan egg masses The surface of molluscan egg masses appears to be a suitable substrate for the settlement of both epizooites and macrophytes. A range of macrofoulers was observed on both leathery egg capsules and gelatinous egg ribbons

Chapter 3 Defence against predation and macrofouling 142 during this study (Table 3.3). This is not surprising considering the surfaces of molluscan egg masses are primarily composed of polysaccharides and proteins (Hunt, 1966; Bayne, 1968; Flower et al., 1969; Karuso, 1986), which could facilitate settlement and attachment (refer to Davis et al., 1989).

The overall incidence of macrofouling on the surface of molluscan egg masses was observed to be low in this study. This could be due to a number of reasons, including chemical defense, the ephemeral nature of the egg masses and parental site selection for the deposition of egg masses.

Macrofauna was more common on the surface of hatched egg capsules

(Table 3.3) suggesting that the colonisation and growth of most fouling organisms is not rapid enough to interfere with the development of most molluscs. Furthermore, most molluscs deposit eggs on the underside of boulders (refer to Chapter 2; Table 2.3), where fouling is unlikely to occur within the time of encapsulation. The fouling of biological surfaces by macroalgae only occurs at sufficiently high levels of illumination (refer to Davis et al., 1989). Consistent with this, algal fouling was only observed on molluscan egg masses that were exposed to sunlight (Table 3.3). In most other cases however, macrofouling may not be a significantly strong selective pressure to influence the evolution of chemical defensive strategies.

3.6 Conclusion

Six potential strategies for reducing the impacts of predation on molluscan egg masses have been identified along the Illawarra Coast, Australia. These include chemical feeding deterrents, physical protection, camouflage or

Chapter 3 Defence against predation and macrofouling 143 cryptic spawn, parental protection, low nutritional quality and rapid embryonic development. Spawning in large aggregated masses may also help reduce predation on molluscan egg masses by providing safety in numbers and restricting predator access to eggs in the centre of the mass. A variety of predators are abundant in the intertidal region and these could have lead to the evolution of multiple defensive strategies. The relative importance of the difference defence mechanisms varies between species and most molluscs probably use a combination of strategies to protect their eggs.

On the other hand, macrofouling appears unlikely to have lead to the evolution of chemical defense in molluscan egg masses, although further studies are required to confirm this. Both gelatinous egg masses and leathery egg capsules were found to provide a potential substrate for a range of fouling organisms. Nevertheless, there was a low frequency of macrofouling on the surface of molluscan egg masses along the Illawarra Coast and fouling was generally heavier on hatched egg capsules. This could be the result of chemical defense, although the short period of encapsulated development for most molluscs may override the need for defence against macrofouling.

Furthermore, many molluscs deposit egg masses on the underside of boulders, which may limit fouling from light dependant species, such as algae.

NOTE

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UNIVERSITY OF WOLLONGONG

Chapter 4:Antimicrobial activity in molluscan egg masses 144

Chapter 4

Antimicrobial activity in molluscan egg masses

4.1 Introduction

4.1.1 The need for novel antibiotics

Contagious disease is a considerable problem to human health and in the agricultural industry. Disease in aquaculture is also predominantly caused by microbially mediated infections (Colwell, 1983) and this has lead to the widespread use of antibiotics overseas (Westra, 1998). Of particular concern is the number of human pathogens have developed multidrug resistance (Cohen,

1992; Neu, 1992; Cannon, 1995; Stinson, 1996) and some bacterial strains are resistant to all existing antibiotics (Setti and Micetich, 1998). There is serious concern regarding new and reemerging infectious diseases for which no effective therapies are available (Stinson, 1996; Cragg et al., 1997; National

Centre for Infectious Diseases, 1997). Prevention and control of these infectious bacteria will require the development of new antimicrobial agents (Cohen,

1992).

The majority of drugs currently in commercial use, and those being developed, are of natural origin (Eisner, 1990; Cragg et al., 1997). Natural products play an invaluable role in the drug discovery process, in relation to all types of diseases.

In particular, 78% of the new antibacterial drugs approved in the U.S. between

1983 and 1994, were drugs of natural origin (Cragg et al., 1997). Nevertheless, only a tiny proportion of the world’s species have been utilised as a source of drugs. The vast diversity of unstudied living organisms provides enormous Chapter 4:Antimicrobial activity in molluscan egg masses 145

resource potential and in particular, marine biological diversity remains relatively untapped (de Vries and Hall, 1994; Flam, 1994; Carté, 1996; Liles, 1996; Cragg et al., 1997; Capon, 1998).

The first attempts to locate antimicrobial activity in marine organisms were initiated around the 1950s (e.g. Burkholder and Burkholder, 1958; Nigrelli et al.,

1959). Since this time, a large number of marine organisms from a broad range of phyla, have been screened for antimicrobial activity (e.g. Shaw et al., 1976;

Rinehart et al., 1981). Many of these organisms have antimicrobial properties, although most of the antibacterial agents that have been isolated from marine sources have not been active enough to compete with classical antimicrobials from microorganisms (Rinehart et al., 1981; Rinehart, 1988). However, by far the majority of marine organisms are yet to be screened and the potential for discovering a useful antibiotic is sufficient to warrant further research (refer to

Liles, 1996).

4.1.2 Antimicrobial activity in molluscs and molluscan egg masses

During two shipboard screening expeditions by Rinehart et al. (1981), a number of molluscs were ranked very high in the priority list of species exhibiting antimicrobial activity. A total of five molluscs were listed in the top fifteen species that inhibited the growth of the bacteria Escherichia coli and Bacillus subtilis. A further seven molluscs were priority listed for activity against the fungus Penicillium atrovenetum and three molluscs were listed for activity against Saccharomyces cerevisiae. Molluscan extracts produced the largest zones of inhibition out of all the marine extracts that were tested against E. coli Chapter 4:Antimicrobial activity in molluscan egg masses 146

and P. atrovenetum. Significantly, different species of mollusc were priority listed for activity in the different assays (refer to Rinehart et al., 1981).

Consequently, marine molluscs may provide potential for isolating compounds with specific activity against certain microorganisms or cell types.

Previous studies on the egg masses of marine gastropods, although few in number, have revealed striking contrasts in the biological activity of the species examined. On one hand, the gelatinous egg masses of molluscs in the

Aplysiidae, exhibit a broad spectrum of biological activity (Kamiya & Shimizu,

1981, Kamiya et al., 1984, 1988, Yamazaki et al., 1984, 1985, Kisugi et al.,

1987, Miyamoto et al., 1988, Yamazaki, 1993, Pennings and Paul, 1991,

Pennings, 1994). Several glycoproteins from the egg masses of Aplysia kurodai and Aplysia juliana were found to have antimicrobial activity (Kamiya et al.,

1984, 1988; Yamazaki et al., 1984, 1985; Kisugi et al., 1987; Yamazaki, 1993).

These egg masses were also found to contain a natural agglutinin capable of agglutinating vertebrate blood cells and marine bacteria (Kamiya and Shimizu,

1981). The egg mass of Aeolidia papaillosa also had weak agglutinating activity against human red blood cells (Rodgers, 1977). Antitumour and antifungal macrolides have been isolated from the egg ribbons of the nudibranch

Hexabranchus sanguineus (Matsunaga et al., 1986, Roesener and Scheuer,

1986). In contrast however, Matsunuga et al. (1986) reported that extracts from the gelatinous egg ribbons of Dendrodoris nigra, were inactive against a number of fungi.

Chapter 4:Antimicrobial activity in molluscan egg masses 147

There appears to be no previous studies reported on the biologically active compounds from leathery egg capsules or the gelatinous egg masses of marine gastropods other than the Opisthobranchs. However, the embryos of the muricids Nucella emarginata and Nucella lapillus, were found to suffer high mortality outside their leathery egg capsules (Pechenik et al., 1984; Rawlings,

1995). Lord (1986) demonstrated that the embryos of Nucella lapillus survived well in sterile seawater to which antibiotics were added, while embryos in unsterile seawater died rapidly outside the capsules. Pechenik et al., (1984) tested the intracapsular fluid of Nucella lapillus egg capsules for antimicrobial activity but found no bacteriostatic properties. However, Lord (1986) demonstrated that the contents of the egg capsules of Nucella lapillus were free from bacterial contamination. The lack of antimicrobial activity reported by

Pechenik et al. (1984) could be due to the use of an inappropriate assay (refer to section 4.1.3). Alternatively, reproductive tracts free of bacteria would be required to produce egg capsules with axenic contents (refer to Lord, 1986).

The Cephalopoda also deposit benthic egg masses, although many

Cephalopods appear to provide their eggs with parental protection (Cousteau and Diole, 1973). It has been noted that the eggs of octopus rapidly develop a fungal infection when the parents are removed (Dr. Lindsey Joll, pers. comm.).

However, the eggs of the common squid Loligo vulgaris are not parentally protected and these were found to contain a natural tranquilliser (Marthy, 1976).

Marthy (1976) suggested that the tranquilliser in the eggs of L. vulgaris provides protection for the young embryos by preventing premature hatching and the Chapter 4:Antimicrobial activity in molluscan egg masses 148

waste of energy from muscular activity. The potential role of these compounds in providing protection from infection has not been investigated.

4.1.3 Assays for detecting antimicrobial activity

Recent collaborations between natural product chemists, molecular pharmacologists, biochemists and cell biologists have lead to sophisticated developments in screening technologies. The focus has moved towards screening compounds in a variety of specific enzyme inhibition and receptor antagonist bioassays (de Vries and Hall, 1994; Carté, 1996). There has also been a large emphasis on discovering drugs that are effective against cancer and HIV (Rinehart et al., 1981; McKee et al., 1997). Several large screening programs have been established by the pharmacological and biotechnology industries (Carté, 1996) and it is difficult for individual researchers to compete with these high throughput screening technologies. However, the value of broad antimicrobial screening should not be underestimated. Screening for antimicrobial activity has proven particularly simple, cost efficient and effective

(Rinehart et al., 1981, Rinehart, 1988; de Vries and Hall, 1994).

There has been no standardisation of the techniques used in screening for antimicrobial activity in marine natural products. However, the assay techniques can influence the results of screening for biological activity (Thompson et al.,

1985, Janssen et al., 1987). Nevertheless, the assay technique is not always reported in studies that state the antimicrobial activity of marine natural products

(e.g. Biskupiak & Ireland, 1983, Hochlowski et al., 1983, Ichiba & Higa, 1986,

Manker & Faulkner, 1986). Some papers report the use of a ‘disk diffusion Chapter 4:Antimicrobial activity in molluscan egg masses 149

assay’ but do not provide any details on the conditions that were used (e.g.

Howchlowski and Faulkner, 1983; Kamiya et al., 1984; Pechenik et al., 1984;

Matsunaga et al., 1986; Roesener and Scheuer, 1986; Bobzin and Faulkner,

1989).

The commonly used assays for detecting antimicrobial activity can be classified according to whether they are performed on solid media (agar) or in liquid media (broth). Both of these types of assays have advantages and disadvantages. However, the detection of antimicrobial activity in all types of assay will depend on the compatibility of the test compound with the media, its solubility and its stability under the test conditions (Spooner and Skyes, 1972;

Janssen et al., 1987). Constituents of the medium may react with components of the test sample and activate or inactivate them. The pH of the medium can also influence the activity of the components or the response of the microorganisms (Spooner and Skyes, 1972; Janssen et al., 1987). The detection of antimicrobial activity will also depend on the temperature, the time of contact with the antimicrobial agent, as well as the number and type of cells

(Spooner and Skyes, 1972). Consequently, all of these factors should be standardized in a screening program.

A test compound or extract will usually only exert an antibacterial action when it is dissolved in an aqueous solution (Spooner and Skyes, 1972). Many of the compounds extracted from natural organisms and a large number of antibacterial compounds do not dissolve well in water. This problem can be overcome by the use of ultrasonication (Janssen et al., 1987), a water miscible Chapter 4:Antimicrobial activity in molluscan egg masses 150

organic solvent (Spooner and Skyes, 1972) or an emulsifying agent (Beylier et al., 1979; Janssen et al., 1987). However, care must be taken to ensure that the final concentration of the solvent or emulsifying agent is not lethal to the microorganism, thus leading to a false positive in the antimicrobial test. A concentration of 5% ethanol and 0.5% of the common emulsifying agent Tween

80, have been found to interfere with cell growth (Spooner and Skyes, 1972;

Masaki et al., 1990). Acetone is thought to be the most suitable organic solvent for solubilizing compounds for antimicrobial testing (Spooner and Skyes, 1972) and this can be used in combination with ultrasonication to dissolve highly lipophilic compounds (e.g. Benkendorff, 1994).

The solid media or agar disk diffusion methods are often the preferred methods for antimicrobial screening in natural extracts (e.g. Thompson et al., 1985;

Janssen et al., 1986; Rinehart, 1988). These assays are relatively quick and simple to perform. However, the relative antimicrobial activity of different samples can not be compared using this assay due to potential differences in physical properties of the antimicrobial agents, such as solubility, volatility and diffusion in agar (Chand et al., 1995). A sharp cut off between bacterial growth and inhibition is not always produced and the zone of inhibition can not be used quantitatively to determine the minimum inhibitory concentration (MIC). Dilutions showing no inhibition in the agar diffusion methods are not comparable to the

MIC values determined using broth dilution methods (Janssen et al., 1986;

Chand et al., 1995). The long incubation time required for the agar diffusion method (18-24hrs) may also render it unsuitable for volatile or unstable antimicrobial agents (Chand et al., 1995). Chapter 4:Antimicrobial activity in molluscan egg masses 151

The lack of bacteriostatic properties found in the egg masses of the gastropod

Nucella lapillus by Pechenik et al., (1984) could be due to the limitations of the agar diffusion method. These researchers placed filter paper disks impregnated with the intracapsular fluid from N. lapillus egg capsules onto agar and failed to detect antimicrobial activity against 13 strains of bacteria. It is possible that no zones of inhibition were detected in this study because the potentially active components did not diffuse from the paper disks onto the agar. This could occur if the active components are highly hydrophobic or insoluble. The lack of antifungal activity found in the egg masses of the nudibranch Dendrodoris nigra

(Matsunaga et al., 1986) may also be due to the use of the disk diffusion assay.

Antimicrobial assays in liquid media are more direct than agar diffusion assays because factors such as diffusion and reaction with agar are avoided (Spooner and Skyes, 1972). However, these assays are generally a lot more time consuming. The inhibition of microbial growth can be observed microscopically or a minimum inhibitory concentration (MIC) can be determined quantitatively.

This may involve plating out a serial dilution (Hattalin et al., 1973) but these methods are laborious and liable to contamination, while the long time period involved can be a major drawback (Chand et al., 1995). MICs can also be determined in broth from the turbidity of the microbial culture (Spooner and

Skyes, 1972). Turbidity can be estimated visually or obtained more accurately by measuring the optical density with a spectrophotometer. However, the solubility and concentration of the test substance could potentially interfere with the turbidity of the microbial culture, thus reducing the accuracy of the MIC. Chapter 4:Antimicrobial activity in molluscan egg masses 152

At least three studies on the antimicrobial properties of marine molluscs have involved the use of turbidometric assays in liquid media (Kamiya et al., 1984;

1988; Yamazaki et al., 1990). Only one study uses a combination of both the disk diffusion assay and a turbidometric assay (Kamiya et al., 1984). These authors reported that the turbidometric assay was rapid, reproducible and useful for quantitative estimation of antibacterial activity. One problem with the turbidometric assays used by Kamiya et al., (1984; 1988) is that it required fairly large amounts of sample (i.e. the assay is performed in ~4.5ml of solution).

Yamazaki et al., (1990) have adapted the turbidometric assay for use in a 96 well plate (i.e. 200µl/well), which is much more suitable for antimicrobial screening projects. Nevertheless, all turbidometric assays are limited by interference from the test samples. This is a particular problem with crude extracts, which are usually incompletely soluble in the aqueous broth.

Recently Chand et al. (1995) developed a liquid assay in 96 well microplates that is appropriate for rapidly screening natural products or extracts for antimicrobial activity. This assay, the fluorescein diacetate (FDA) assay is reported to be an accurate and reproducible method for determining the MIC of crude extracts and purified compounds (Chand et al., 1995). The method is economical in time and resources making it well suited for use in screening programs, as well as for bioassay directed purification of active compounds.

The assay depends on the activity of non-specific esterases, which are produced by metabolically active microorganisms. These metabolic enzymes have been shown to hydrolyse colourless, non-fluorescent fluorescein diacetate Chapter 4:Antimicrobial activity in molluscan egg masses 153

(FDA) to fluorescein, a yellowish green compound, which fluoresces under UV light (Lundgren, 1981; Chand et al., 1995). Thus the viability of a microbial culture can be determined based on a colour change or by the detection of fluorescence. This overcomes the problems of interference from the test compounds in typical turbidometric assays and it is easy to control for autofluorescence.

Most microorganisms produce non-specific esterases (Lundgren, 1981). Thus the FDA assay is suitable for use with a wide range of different microorganisms.

The method has been used previously to detect antimicrobial activity against a range of human pathogens in essential oils, crude extracts from ants and marine natural products (Benkendorff, 1994; Chand et al., 1995). The method has not been tested on marine pathogens, although marine microalgae have been shown to hydrolyse FDA (Gilbert et al., 1992). Significantly, only four previous studies on molluscan metabolites have tested for antimicrobial activity against marine pathogens (Kamiya et al., 1984; Pechenik et al., 1984; Manker and Faulkner, 1986; Kamiya et al., 1988).

It is appropriate to distinguish between bacteriostatic activity and bactericidal activity. Bacteriostasis is applied to the action of a chemical antagonist on a microorganism under conditions where growth can normally occur (Spooner and Skyes, 1974). On the other hand bactericidal activity refers to the situation where a microorganism subjected to the action of a chemical antagonist is unable to recover (Spooner and Skyes, 1974). Both the disk diffusion assay and the broth methods can only be used to detect bacteriostatic activity. Bactericidal Chapter 4:Antimicrobial activity in molluscan egg masses 154

activity may be detected by examining the cells under a microscope or more accurately by incubating the culture in fresh media and assessing the ability of the cells to recover. The latter method was used in conjunction with the FDA assay by Benkendorff (1994), to assess the antimicrobial properties in the exocrine secretions of ants. This combination of methods was found to be useful for producing an accurate and reproducible overall assessment of antimicrobial activity.

Another potential problem associated with the detection of antimicrobial activity from natural sources is related to the preparation and storage of material.

Reinhart et al. (1981) found that many active marine compounds decomposed during storage, even when the specimens were frozen. The presence of many biologically active compounds may also vary temporally (de Vries and Hall,

1994). Significantly, the antimicrobial activity in the egg mass of the sea hare

Aplysia juliana was found to disappear over the period of larval development

(Kamiya et al., 1988). Consequently, a single collection of an organism may not necessarily reflect its potential to provide useful chemicals. Similar concerns have been described for essential oils from plants (Janssen et al., 1987).

The solvents and methods used to extract the samples will also influence the type of compounds that are recovered and could therefore affect the likelihood of detecting antimicrobially active compounds from organisms. An interesting example of this problem is provided by Eisner et al., (1970), who reported that the egg capsules of millipedes had no antibacterial or antifungal substances.

However, the extraction procedure used by these researchers would have only Chapter 4:Antimicrobial activity in molluscan egg masses 155

permitted the recovery of hydrophilic components. Consequently it remains possible that the millipede eggs are chemically protected by hydrophobic antimicrobial compounds. Shimizu (1985) reported that the isolation of compounds from marine sources is heavily in favour of lipid soluble compounds because the isolation and purification of water soluble compounds is typically more difficult. However, more recently a number of studies have concentrated on hydrophylic compounds (e.g. Kisugi et al. 1987; Yamazaki et al., 1990).

General screening for antimicrobial activity should ultimately use a solvent system that will permit the extraction of both hydrophilic and hydrophobic components.

An additional problem associated with the extraction of natural products is that biologically active constituents could be lost or degraded during the process.

Some compounds may react with the solvent or with oxygen (de Vries and Hall,

1994). Many volatile or heat sensitive components could also be lost during the process of evaporating the solvent (Shimizu, 1985). Even mild procedures such as lyophilization can result in the loss of biological activity (de Vries and Hall,

1994). These problems could be overcome by using an antimicrobial screen that uses biological samples directly, without extraction. Solid or semisolid samples can be placed directly onto the surface of agar and tested for antimicrobial activity in a zone of inhibition assay (Spooner and Skyes, 1972).

The applicability of this method to marine specimens has not been tested previously. However, this method will be limited by the same factors that limit the disk diffusion assay. Ultimately, a combination of methods would be the best way to assess the antimicrobial potential of biological organisms. Chapter 4:Antimicrobial activity in molluscan egg masses 156

4.1.4 Microfouling and symbiosis

The importance of epibiosis or symbiosis to the success of encapsulated marine embryos has received little attention. Bacteria have been found to rapidly colonise inanimate surfaces in the ocean (Zobell and Allan, 1935; Wahl et al.,

1994). The egg masses of marine molluscs are composed primarily of proteins and polysaccharide (Hunt, 1966; Flower et al., 1969; Karuso, 1987) making them highly suitable for the settlement of microorganisms. The egg ribbons of the nudibranch Archidoris montereyensis were shown to be a favourable habitat for numerous species of diatoms (Biermann et al., 1992). By comparison the egg ribbons of the cephalspidean gastropod, Haminoea vesicula, were much less vulnerable to microalgal fouling (Biermann et al., 1992). Different taxa may be differentially equipped with devices to prevent microbial fouling on the surface of their egg masses.

Microalgal fouling on the egg ribbons of the nudibranch Archidoris montereyenis was shown to cause significant embryonic mortality (Biermann et al., 1992). The negative effect of epibiosis to the embryos can probably be attributed to the addition of wastes to the egg mass or the depletion of oxygen. Microbial epibionts on the eggs of marine invertebrates are thought to result in asphyxia

(Austin, 1988). Microorganisms fouling the surface of the egg masses from two opisthobranch gastropods and a polychaete were found to play a large role in the production and consumption of oxygen (Cohen and Strathmann, 1996).

Epibiotic microorganisms could also form symbiotic relationships with the eggs of some marine species. Microorganisms that have settled onto a surface can Chapter 4:Antimicrobial activity in molluscan egg masses 157

play an important role in inhibiting further settlement (Davis et al., 1989). A symbiotic marine bacterium, found on the surface of shrimp eggs, has been shown to produce a compound that protects the embryos from fungal infection

(Gil-Turnes et al., 1989). Similarly, an unidentified bacterium covering the surface of American lobster eggs was found to produce large amounts of a phenolic compound, which could effectively inhibit pathogenic microorganisms

(Gil-Turnes and Fenical, 1992). It is possible that symbiotic microorganisms protect the egg masses of marine molluscs in a similar manner. Ultimately, symbiotic microorganisms have been found in association with a range of marine organisms and many of these produce biologically active compounds

(Austin, 1992; Kobayashi and Ishibashi, 1993).

Microorganisms continue to be an important source of pharmaceutical agents and marine bacteria offer a diverse and relatively new source for drug development (Fenical, 1993). Consequently, investigation of the microorganisms associated with the surface of molluscan egg masses could provide an important pharmaceutical lead. Microorganisms are generally easier to maintain and culture than macroorganisms (Holmström and Kjelleberg,

1994). There is also some evidence that antifouling agents from sessile marine organisms have antibacterial properties (Al-Ogily and Knight-Jones, 1977; Walls et al., 1993; Koh, 1997). Marine organisms that are free from microbial fouling may also be useful in the development of novel antifouling technologies (refer to

Holmström and Kjelleberg, 1994).

Chapter 4:Antimicrobial activity in molluscan egg masses 158

4.2 Objectives

The objectives of this component of the study were to screen the egg masses of a broad range of marine molluscs for antimicrobial activity. The egg masses of two freshwater molluscs, one terrestrial mollusc and four polychaetes were also tested to provide a preliminary assessment of whether antimicrobial activity was likely to be a general property of invertebrate egg masses. The egg masses of many molluscs are small, difficult to collect and/or rare (refer to Chapter 2).

Consequently, there was a need to optimise the assays to detect antimicrobial activity in minimal quantities of material. The egg capsules of Dicathais orbita and the egg masses of Aplysia juliana were used to refine two antimicrobial assays: the zone of inhibition assay and the fluorescein diacetate assay. The egg masses of these species are seasonally abundant and representative of the two major forms of molluscan egg masses (i.e leathery capsules and gelatinous ribbons).

The egg masses from 43 molluscs and four polychaetes were then screened for antimicrobial activity against three bacteria (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa), which are routinely used for testing disinfectants (Olsen, 1979). These are all human pathogens that have developed some resistance to common antibiotics, particularly in the clinical environment. In addition, the eggs of Aplysia juliana and Dicathais orbita were tested for activity against the yeast Candida albicans and several marine pathogens. Candida albicans is an important minor human pathogen that often does not respond to conventional antibiotic therapy (Odds, 1988). The marine bacteria included Vibrio anguillarum, which is a world wide fish and molluscan Chapter 4:Antimicrobial activity in molluscan egg masses 159

pathogen (Austin, 1988), Vibrio harveyi and Vibrio alginolyticus, which are pathogens of fish, molluscs and crustaceans (Austin, 1988; Sutton and Garrick,

1993; Koh, 1997), as well as the fish pathogen Enterococcus sericolicida

(Jeremy Carsons; pers. comm.). Vibrio alginolyticus also occurs in bacterial films and may play a role in the initial stages of surface fouling (Koh, 1997).

Preliminary observations were also made on potential microfoulers or symbiotic microbes on the surface of the molluscan egg masses.

4.3 Methods

4.3.1 Collection and preparation of egg material

The egg material from 35 intertidal, five estuarine, two fresh water and one terrestrial mollusc, as well as four polychaetes were collected for antimicrobial testing (Table 4.1). Most of the specimens were collected along the Wollongong coast, N.S.W., Australia (Chapter 2, Figure 2.1). However, the egg masses from three gastropod species and one polychaete were collected from the

Mediterranean Sea, Spain. The eggs of two fresh water gastropods were collected from laboratory cultures and the eggs of a terrestrial snail were collected from a terrarium. Egg masses from many of the Opisthobranchs were deposited in aquaria shortly after collection of the adult from the field. A summary of all the species tested, the site of collection and the amount collected is provided in Table 4.1. Voucher specimens are lodged in the

Department of Biological Sciences, University of Wollongong.

Most of the egg masses were collected shortly after being laid and were used immediately or placed in the freezer (-200C) until used. Antimicrobial activity Chapter 4:Antimicrobial activity in molluscan egg masses 160

was assessed at different developmental stages for a subset of eight species

(Table 4.1, 4.2). The developmental stage was determined by changes in colouration or firmness of the egg masses (Table 4.2). The developmental stage was confirmed by inspection of the embryos or veligers under a dissecting microscope. An open aperture at the top of leathery egg capsules also indicated the commencement of veliger hatching. The empty leathery egg capsules from Dicathais orbita and Mitra carbonaria were also collected for testing in the zone of inhibition assay.

The egg masses of most species were ground with a mortar and pestle prior to use in the zone of inhibition assay. Some were tested intact by cutting a strip of egg mass with sterile forceps. For the FDA assay the egg masses were blended in solvent using a Waring blender or cut open using a pair of scissors. Organic extracts were prepared by soaking macerated egg masses in organic solvent.

Chloroform/methanol (1:1, v:v) was used for most species but additional extracts were prepared from the egg masses of Aplysia juliana, Dicathais orbita and Conuber c.f. sordidus using ethanol and/or diethyl ether. The solvent was decanted and replaced after three hours and then after a further six hours. The sample was then left to soak overnight and then the fractions were combined and filtered. A small volume of water was added to the extract when necessary to induce the formation of two layers. The chloroform or ether layers were separated from the water/ methanol layers in a separating funnel. The solvent was then removed by rotary evaporation under vacuum. The dried extract was transferred to a small weighed vial by sequential resuspension in small volumes

(~0.5ml) of methanol (MeOH) and dichloromethane (DCM). The bulk of the Chapter 4:Antimicrobial activity in molluscan egg masses 161

Table 4.1: Species of molluscs (arranged by family) and polychaetes that were screened for antimicrobial activity in their egg masses. The developmental stage refers to the egg mass unless recorded as adults. All species are from the intertidal area on the Wollongong Coast, N.S.W, unless otherwise stated. Where the egg masses were collected from aquaria, the site at which the adults were collected is noted in parentheses. The amount of material collected for antimicrobial testing is recorded per egg mass for species that were only tested in the zone of inhibition assay but is provided in grams for material that was solvent extracted.

Family Species Developmental Site collected Amount stage collected Neritopsidae Nerita atramentosa Unknown Bellambi 25 egg capsules Littorinidae Bembicium nanum Freshly laid Austinmer 161.5g North Wollongong 47.6g Wombarra 12g Naticidae Conuber cf. sordidus Freshly laid St. Georges Basin 900g Lake Illawarra 150g Ranellidae Cabestana spengleri Unknown Towradgi 15g Muricidae Dicathais orbita Freshly laid Towradgi 292g Flagstaff Hill 115g Hatching Towradgi 64.8g Empty capsules Towradgi 20 capsules Adults Towradgi 2 (108.1g) Agnewia tritoniformis Freshly laid South Shellharbour 10.5g Hatching South Shellharbour 8 capsules Morula marginalba Freshly laid Bellambi 0.26g Bedeva hanleyi Freshly laid Coledale 1.56g Lepsiella reticularis Freshly laid Towradgi 1.2g Trunculariopsis Unknown Punta Santa Ana, 1 egg mass trunculus Blanes, Spain (8.2g freeze- dried) Ceratostoma erinaceum Unknown Sitges, Spain 30 capsules (1.2g freeze- dried) Adults Sitges, Spain 1 specimen Mitridae Mitra carbonaria Freshly laid South Shellharbour 6.4g + 10 capsules Well developed South Shellharbour 10 egg capsules Empty capsules South Shellharbour 10 capsules Mitra boudi Well developed South Shellharbour 25 egg capsules Conidae Conus paperliferus Freshly laid Bellambi 4.3g + 6 capsules Hatching Bellambi 10 capsules Unknown Unidentified sp. 2 Unknown Shellharbour 8 egg capsules Amphibolidae Salinator fragilis Freshly laid Lake Illawarra 25.4g Adults Lake Illawarra 6 specimens Salinator solidus Unknown Towra Point 52.9g Adults Towra Point 24g Siphonariidae Siphonaria denticulata Freshly laid Austinmer 12.3g Wombarra 15g Well developed Austinmer 12.6g Siphonaria zelandica Freshly laid Wombarra 5.6g Planorbidae Isidorella hainesi Freshly laid Aquaria (Lithgow) 28.8g Well developed Aquaria (Lithgow) 3g Glyptophysa gibbosa Freshly laid Aquaria (Lithgow) 8 egg masses Camaenidae Meridolum gulosum 1 week Aquaria (Figtree) 6 egg capsules Unknown Unidentified sp. 14 Unknown Puckeys creek 6 egg ribbons Aplysiidae Aplysia juliana Freshly laid Bulli Pool 670g Well developed Bulli Pool 104.9g Chapter 4:Antimicrobial activity in molluscan egg masses 162

Table 4.1 continued.

Family Species Developmen Site collected Amount tal stage collected Aplysiidae Aplysia sydneyensis Freshly laid Bellambi 11.7g Aplysia parvula Freshly laid Wombarra Pool 1 egg ribbon (2g) Aplysia dactylomela Unknown Clovelly 1 egg ribbon Stylocheilus Unknown Bulli Pool 30.6g longicauda Dolabrifera dolabrifera Unknown Bellambi ~ 1/6 egg ribbon Philinidae Philine angasi Freshly laid Bulli Pool 178.4g Pleurobranchidae Pleurobranchus peroni 1 week old Aquaria (Bellambi) 1 egg ribbon Pleurobranchea sp. Freshly laid Aquaria (Bellambi) 2 egg ribbons Bullinidae Bullina lineata Freshly laid Aquaria (Shellharbour) 2 egg ribbons Hydatinidae Hydatina physis Freshly laid Aquaria (Bulli) 1 egg ribbon Oxynoidae Oxynoe viridis Unknown Bellambi 1 egg ribbon Dendrodorididae Dendrodoris fumata Freshly laid Aquaria (Bellambi) 2 egg ribbons Dorididae Platyodoris galbannus Freshly laid Aquaria (Bellambi) 2 egg ribbons Jorunna pantherina Freshly laid Aquaria (Shellharbour) 1 egg ribbon Goniodorididae Goniodoris sp. Unknown Towradgi 4 egg ribbons Polyceridae Plocampherus imperialis Freshly laid Aquaria (Towradgi) 1 egg ribbon Glaucidae Spurilla neopolitana Unknown Sitges, Spain 1.41g freeze-dried Adults Sitges, Spain 4 individuals 0.67g freeze-dried Unidentified sp. 13 Unknown Bass Point 1 egg ribbon Loliginidae Sepioteuthis australis Unknown Coledale beach 1 egg mass, 77g Towradgi pool 1/5 egg mass POLYCHAETA Eupolymnia sp. Unknown Punta Santa Ana, 1.8g freeze-dried Blanes, Spain Unidentified sp. 1 Unknown Bulli Pool 6 egg masses Unidentified sp. 2 Unknown Bulli pool 2 egg masses Unidentified sp. 3 Unknown Lake Illawarra 72g

Table 4.2: The primary characteristics used to determine the stage of development in the egg masses of eight molluscs.

Species Fresh egg mass Well developed egg mass Agnewia tritoniformis Yellow intracapsular fluid Purple intracapsular fluid

Dicathais orbita Yellow intracapsular fluid Purple intracapsular fluid

Mitra carbonaria White intracapsular fluid Black intracapsular fluid

Mitra boudi White intracapsular fluid Black intracapsular fluid

Conus paperliferus Large yellow eggs Shelled veligers and aperture open on capsules Siphonaria denticulata Egg mass firm and white Egg mass brown and soft

Isidorella hainesi Small white eggs Shelled veligers

Aplysia juliana Firm jelly with yellow eggs Soft jelly with brown veligers

Chapter 4:Antimicrobial activity in molluscan egg masses 163

remaining solvent was removed under a stream of nitrogen gas and the last traces by evaporation in vacuo (0.1mm Hg; 30min; room temperature).

Chloroform extracts from the egg masses were fractionated by flash silica

(Merck; 60 mesh) chromatography. The extracts were passed through a small silica column (6cm length; 1cm diameter) using redistilled DCM. Two fractions were collected and then 10% MeOH in DCM (redistilled) was used to wash the column and the final fraction was collected. The fractions were dried under a stream of nitrogen and stored at -200C until required.

The adult tissue from several species of mollusc was also tested for antimicrobial activity (Table 4.1). The adults were killed in the freezer and then removed from their shells using a vice. The tissue was ground using a mortar and pestle, then extracted in chloroform/ methanol (1:1, v:v), as detailed above.

Extracts from seawater, estuarine mud and intertidal pebbles were also prepared using the same procedure and tested for antimicrobial activity in the

Fluorescein diacetate assay.

4.3.2 Maintenance and preparation of microbial cultures

Stock cultures of Candida albicans ACM4581, Escherichia coli ACM845,

Staphylococcus aureus ACM844 and Pseudomonas aeruginosa ACM846 were obtained from the Culture Collection at the University of Queensland and maintained at –780C in 15% glycerol. Additional cultures of C. albicans

(AMMRL 36.42, AMMRL 36.70) were obtained from the Australian National Chapter 4:Antimicrobial activity in molluscan egg masses 164

Reference Laboratory in Medical Mycology, the Royal North Shore Hospital,

Sydney. These were maintained in 0.85% saline according to Muir (1988).

The cultures were prepared by streaking onto Saboraud dextrose agar (SDA)

(DIFCO 0109-17-1; pH 5.6) for C. albicans and Nutrient Agar (NA) (Oxoid CM3; pH 7.4) for E. coli, S. aureus and P. aeruginosa. After an overnight incubation, single colonies were used to innoculate sterile liquid medium. The broth for the three bacteria consisted of yeast extract (5 g, ICN 103303-17), peptone (10 g,

Oxoid L37) and NaCl (5 g, ICN 102892) in distilled water (100 ml). Sabouraud liquid medium (Oxoid CM147, pH 5.7), (3 g in 100 ml distilled water) was used for C. albicans. Innoculated broths were placed on an orbital shaker (150 rev/min) and incubated overnight (370C). Two additional broths were trialed for

C. albicans in the FDA assay. These consisted of peptone (1 g, Oxoid L37) and glucose (4g SIGMA G-8270) in distilled water (100 ml) or malt extract (0.3 g,

Oxoid L39), yeast extract (0.3 g), 0.5g peptone (0.5g) and glucose (1 g) in distilled water (100 ml).

Five marine pathogens Enterococcus sericolicida, Vibrio anguillarum, Vibrio alginolyticus, Vibrio harveyi and Vibrio tubiashi, were provided by the

Tasmanian Department of Agriculture and Fisheries. Cultures were prepared by streaking onto Marine Agar (Difco 2216, pH 7.6) and incubating overnight at

250C. After an overnight incubation single colonies were used to inoculate sterile liquid medium (pH 7.5) composed of Nutrient broth (2.5 g, Oxoid No. 2), yeast extract (0.3 g) and NaCl (5 g) in distilled water (100 ml). Innoculated broths were placed on an orbital shaker (150 rev/min) and incubated overnight Chapter 4:Antimicrobial activity in molluscan egg masses 165

at 250C. The freeze-dried culture of Vibrio tubiashi did not recover after streaking onto marine agar. An attempt was made to recover the culture by directly inoculating the freeze-dried cells into liquid medium. The culture recovered after three days of incubation at 250C but inspection under the light microscope revealed a mixed culture dominated by gram positive bacteria

Coryebacterium sp., Lactobacillus sp. and Staphylococci sp. (i.e. typical skin flora). There were some gram negative bacteria present that were likely to be

Vibrio sp. but it was not possible to determine if these were Vibrio tubiashi or contaminants from the other Vibrio cultures. This was the only contaminated culture that was obtained during the course of the study and was only used to provide further information on the antimicrobial properties of Dicathais orbita egg masses.

4.3.3 Zone of inhibition assay

This assay is a modification of the traditional disk diffusion assay described by

Bauer et al., (1966). Overnight cultures of the microorganisms were diluted to an absorbance of 0.08-0.12 (600nm) and grown to 0.2 (~ 40min on orbital shaker) in 25 ml Macartney bottles. Candida albicans was diluted to 0.16 and then grown to an absorbance of 0.22-0.24. The absorbance was measured on an Ultrospec 2000 spectrophotometer (Pharmacia Biotech). The exponentially growing cultures were then pipetted onto the agar and evenly spread across the surface. Several volumes of culture were trialed to obtain the most consistent lawn. These ranged from 50 l to 1ml of culture, with the excess culture liquid removed with a pipette after spreading. It was found that flooding the surface of the agar with 1ml of culture gave the most even lawn of bacteria. Duplicate Chapter 4:Antimicrobial activity in molluscan egg masses 166

samples of the egg material were placed onto the microbial lawn using forceps sterilised in ethanol and flamed. The plates were incubated overnight at 370C for human pathogens and 250C for marine pathogens before the plates were photographed and zones of inhibition recorded.

In general, duplicate samples of egg material were placed on each agar plate.

Replicate plates were prepared for the egg capsules of Dicathais orbita, Mitra carbonaria and Agnewia tritoniformis. Replicate plates were also prepared for the gelatinous egg masses of Bembicium nanum, Aplysia juliana, Aplysia sydneyensis and Stylocheilus longicauda. Different preparations of the egg material from several species were placed in duplicate on the same plate for direct comparison. These included crushed and intact egg masses, as well as fresh, freeze-dried and frozen samples of egg mass. The intracapsular fluid was compared to the intact and crushed egg capsules from Dicathais orbita. The different developmental stages of the egg masses from several species were also compared in duplicate on the same plate. All of the different preparation methods were replicated at least twice for each microorganism with samples of

Dicathais orbita and Aplysia juliana egg masses. Alternative methods for the zone of inhibition assay were trialed using the egg ribbons of Aplysia juliana and

Dicathais orbita and these are outlined in Appendix 4.1.

4.3.4 The Fluorescein Diacetate (FDA) assay

The egg extracts were tested for antimicrobial activity using the FDA assay described by Chand et al., (1995). Overnight cultures of the microorganisms were diluted to an absorbance of 0.12 (600nm) and grown to 0.18 (ca 30min; Chapter 4:Antimicrobial activity in molluscan egg masses 167

370C or 250C for marine microbes; 150 rev/min). The wells of a tissue culture plate (3072 Microtest III, Becton Dickinson) were filled with 175 l of the culture.

To each well, 20 l of the test substance or appropriate control was added.

Three replicates were made at each test concentration. The microtiter plate was incubated for 30 min at 370C (or 250C for marine microbes) before 5 l

FDA (0.2% solution in acetone) was added. Incubation was continued for a further two hours or until the production of fluorescein was easily visible under an ultraviolet light (λ 254 nm). The results were recorded as positive or negative according to the detection of fluorescence. FDA hydrolysis was also measured spectrophotometrically. The absorbance of the microbial cultures was measured at 490nm referenced to 600nm on a Spectra Max 250 (Molecular Devices).

However, inconsistent results were obtained from the spectrophotometric measurements due to interference from the colour and insolubility of the test samples.

Organic extracts used in the assay were dissolved in acetone and water/methanol extracts were dissolved in 20% (by volume) acetone in MilliQ water. The extracts were vortexed prior to use and extracts that had not completely dissolved were placed in an ultrasonic bath (1 min). All extracts were tested at a concentration of 0.1, 1 and 10 mg/ml unless otherwise stated.

Fractions from the chloroform extract were tested at 0.01, 0.1 and 1 mg/ml. The egg masses from five species were extracted on at least two separate occasions for testing in the FDA assay. These species were Dicathais orbita

(fresh and hatching egg capsules), Aplysia juliana, Bembicium nanum, Conuber c.f. sordidus, Siphonaria denticulata and Sepioteuthis australis. The different Chapter 4:Antimicrobial activity in molluscan egg masses 168

extracts were tested in separate assays with fresh cultures of the microorganisms.

Three replicate controls consisting of 20 l of acetone and 20 l of Milli Q water, both with FDA, were added to each test plate to determine any affects of the acetone on the viability of the cells. Additional controls included 20 l of test substance in 175 l of broth with FDA, to ascertain whether the test compound hydrolysed FDA, and 195 l of broth with FDA to check for contamination in the broth. Additional controls used in some assays involved large volumes of dried down solvent, prepared in the same way as the egg extracts. Samples of seawater and estuarine mud were also extracted and tested for antimicrobial activity in the FDA assay. Additional details on the development of methods and alternative techniques for the FDA assay are provided in Appendix 4.2.

4.3.5 Antimicrobial (cell lysis/ cell stasis) assay

After the FDA assay was completed, 20µl of culture (four replicates) from all the wells that did not show fluorescence, were spread on to agar to determine if the cells could recover. The plates were incubated overnight at 370C for human pathogens and 25 0C for marine microbes. Counts of visibly growing colonies were performed and compared to a dilution series of a control culture from the

FDA plate containing acetone. Chapter 4:Antimicrobial activity in molluscan egg masses 169

4.3.6 Microfouling and symbiosis

The egg masses from a range of marine molluscs were placed on Marine Agar

(Difco 2216) with sterile forceps and incubated at 250C for up to one week. The area surrounding the masses was checked daily for microbial growth. The plates used in the zone of inhibition assay were also checked for the growth of foreign microorganisms. Any evidence of competitive inhibition of the test pathogen in the area surrounding the eggs was noted. Plates containing samples of Dicathais orbita and Aplysia juliana egg masses were kept refrigerated (40C) for one week and observed again for microbial growth on or around the eggs. Samples of estuarine mud and intertidal sand were also tested for foreign microbial growth.

4.4 Results

4.4.1 Antimicrobial activity in benthic invertebrate egg masses The freshly laid egg masses from 42 molluscs and 4 polychaetes were tested for antimicrobial activity in the Zone of Inhibition (ZI) and/or the Fluorescein

Diacetate (FDA) assay and 36 of these species effectively inhibited the growth of at least one test microorganism (Table 4.2). Species from 19 out of 23 families of mollusc were found to have antimicrobial properties in their egg masses (Table 4.2). These included estuarine (Naticidae & Amphibolidae), fresh water (Planorbidae) and terrestrial species (Camaenidae), as well as marine species from two classes (Gastropoda and Cephalopoda).

Antimicrobial activity was detected in the egg masses from nine species in the

FDA assay but not in the Zone of Inhibition assay (Table 4.2). A further six Chapter 4:Antimicrobial activity in molluscan egg masses 170

species did not show activity in the Zone of Inhibition assay but were not tested in the FDA assay (Table 4.2). The egg mass from one species of Mitridae clearly inhibited microbial growth in the Zone of Inhibition assay but extracts from this egg mass did not inhibit FDA hydrolysis (Table 4.2). Notably, the extract from this species rapidly oxidised to produce an insoluble black substance.

4.4.2 Antimicrobial properties at different stages of development

The egg masses from seven species of mollusc were tested for antimicrobial activity at two different stages of development (freshly laid and hatching). The fresh egg masses from all of these species inhibited the growth of at least one test microorganism, in at least one antimicrobial assay (Table 4.3). However, none of the hatching egg masses were found to completely inhibit the growth of any test microorganisms in either assay (Table 4.3). For example, large consistent zones of inhibition were produced around the freshly laid eggs of

Aplysia juliana, whereas only small and inconsistent inhibition areas were observed around the well-developed eggs of this species (Figure 4.1). The hatching egg mass of Mitra boudi also did not inhibit microbial growth, although the fresh egg mass of this species was not tested (Table 4.3). The empty egg capsules of Dicathais orbita, Mitra carbonaria and Conus paperliferus also showed no antimicrobial activity in the Zone of Inhibition assay.

Samples of adult tissue from five species of mollusc were also tested for antimicrobial activity. The adult tissue from only one of these species (Spurilla neopolitana) clearly inhibited microbial growth (Table 4.3). However, the adult Chapter 4:Antimicrobial activity in molluscan egg masses 171

Table 4.2: The proportion of freshly laid invertebrate egg masses found to have antimicrobial activity against at least one microorganism in the Zone of Inhibition (ZI) and Fluorescein Diacetate (FDA) assays. The classification of molluscs is based on Beesley et al., 1998. CLASS/ Order/ Family Proportion of species active Superorder Infraorder ZI FDA Total PHYLUM: MOLLUSCA GASTROPODA Neritopsina Neritodea Neritidae 0/1 0/1 Caenogastropoda Littorinimorpha Littorinidae 0/1 1/1 1/1 Naticidae 0/1 1/1 1/1 Ranellidae 1/1 1/1 Neogastropoda Muricidae 3/4 7/7 7/7 Mitridae 1/1 0/1 1/1 Conidae 1/1 1/1 1/1 Unidentified 1/1 1/1 Heterobranchia Basommatophora Amphibolidae 0/2 2/2 2/2 Siphonariidae 1/1 2/2 2/2 Planorbidae 2/2 1/1 2/2 Unidentified 0/1 0/1 Eupulmonata Camaenidae 1/1 1/1 Anaspidae Aplysiidae 5/6 3/3 6/6 Cephalaspidea Philinidae 0/1 1/1 1/1 Notaspidea Pleurobranchidae 1/2 1/2 Saccoglossa Oxynoidae 1/1 1/1 Nudibranchia Dendrodorididae 2/3 2/3 Gonidorididae 0/1 0/1 Polyceridae 0/1 0/1 Glaucidae 0/1 1/1 1/1 Unidentified 1/1 1/1 CEPHALOPODA Debrachia Teuthoidae Loliginidae 0/1 1/1 1/1 PHYLUM: ANNELIDIA POLYCHAETA Unidentified 1/4 2/2 2/4 TOTAL INVERTEBRATE EGG MASSES 20/41 24/25 36/46 Chapter 4:Antimicrobial activity in molluscan egg masses 172

Table 4.3: The antimicrobial activity of 12 molluscs at tested at different stages of development in the Zone of Inhibition (ZI) and Fluorescein Diacetate (FDA) assays. + indicates the specimen unambiguously inhibited the growth of at least one microorganism in the given assay; – indicates the specimen showed no clear antimicrobial properties.

Species Developmental stage Fresh Hatching Adults egg mass egg mass ZI FDA ZI FDA ZI FDA Agnewia tritoniformis + + - - Dicathais orbita + + - - - - Ceratostoma erinaceum + + - Mitra carbonaria + - - - Mitra boudi - Conus paperliferus + + - Salinator fragilis - + - Salinator solidus - + - - Siphonaria denticulata + + - - Isidorella hainesi + + - - Aplysia juliana + + - - Spurilla neopolitana - + - + Proportion active 8/11 10/11 0/8 0/6 0/5 1/3

Figure 4.1: The antimicrobial activity of the fresh (left) and well developed (right) egg ribbons from Aplysia juliana, tested against Escherichia coli on nutrient agar. Chapter 4:Antimicrobial activity in molluscan egg masses 173

tissue from two species was not tested in the Fluorescein Diacetate assay and the egg mass from one of these species (Salinator fragilis) did not show activity in the Zone of Inhibition assay. All of the adult tissue that was tested on agar in the Zone of Inhibition assay was contaminated with the growth of foreign (non- test) microorganisms.

4.4.3 Zone of Inhibition assay

The zone of inhibition assay was useful for detecting antimicrobial activity in invertebrate egg masses against a range of human and marine pathogens.

Clear zones of inhibition could be detected with at least one egg mass against the Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa, the

Gram positive bacterium Staphylococcus aureus, and three Vibrio spp., as well as the yeast Candida albicans. However, zones of inhibition were difficult to detect against Enterococcus sericolicida because this bacterium did not produce a readily visible ‘lawn’ on the agar plates. Consequently, the results against this marine bacterium in the Zone of Inhibition assay are subjective.

A clear zone of inhibition against at least one human pathogen was observed with the egg masses of 21 species of mollusc and one polychaete (Table 4.2,

4.4). The egg ribbons of Aplysia juliana produced the largest zones of inhibition against all the test organisms. Large zones of inhibition were observed with pieces of A. juliana egg ribbons that were less than 1cm in length. Inconclusive results were obtained with the egg masses of five molluscs and two polychaetes

(Table 4.4). These egg masses produced small areas where microbial growth was inhibited but a consistent zone of inhibition was not observed. There was Chapter 4:Antimicrobial activity in molluscan egg masses 174

no evidence of antimicrobial activity in the egg masses of ten molluscs and one polychaete using the zone of inhibition assay. These specimens were contaminated with foreign microbial growth surrounding the egg material (Table

4.4; e.g. Fig. 4.2).

Crushing the egg masses greatly increased the chance of detecting a zone of inhibition. Zones of inhibition were detected against more of the microorganisms when the samples were crushed and the zones of inhibition were generally larger. For example, the crushed egg ribbons of Aplysia juliana produced large zones of inhibition (>1cm) against all four human pathogens but minimal inhibition was observed around the eggs that were not crushed (e.g. Figure

4.3). The whole crushed egg capsules of Dicathais orbita showed similar antimicrobial activity to the intracapsular fluid (Table 4.4). However, a more consistent zone of inhibition surrounded the intracapsular fluid. Leathery egg capsules that were not crushed produced no zones of inhibition. The egg ribbons of five species of Opisthobranchs that showed no zone of inhibition were not crushed for the assay (Table 4.4).

Samples of Aplysia juliana eggs were shown to retain their antimicrobial activity after being frozen or freeze dried (Table 4.4). Zones of inhibition were similar for the fresh and frozen eggs, but smaller with the freeze-dried eggs. The freeze- dried egg capsules of Dicathais orbita did not produce a clear zone of inhibition in comparison to the fresh crushed egg capsules (Table 4.4). The freeze dried egg masses of Trunculariopsis trunculus and Spurilla neopolitana did not show any antimicrobial activity but no fresh samples were available for testing. The Chapter 4:Antimicrobial activity in molluscan egg masses 175

freeze-dried egg capsules of Ceratostoma erinaceum showed minimal activity against one bacterium but produced inconclusive results against the other two test microorganisms (Table 4.4). Freeze drying removed the contamination that was observed around the fresh eggs of Bembicium nanum but these egg still did not show any antimicrobial activity (Table 4.4).

The Gram positive bacterium Staphylococcus aureus was the most susceptible to components of the molluscan egg masses. The growth of S. aureus was inhibited by the egg masses of 18 species (Table 4.4) and the largest zones of inhibition were consistently produced against this bacterium. The Gram negative bacterium Escherichia coli was inhibited more often than the other

Gram negative bacterium Pseudomonas aeruginosa. The yeast C. albicans

(ACM4581) showed the most resistance to inhibition by the molluscan egg masses and was only completely inhibited by the egg ribbons of Aplysia spp.

(Table 4.4). The egg ribbons of Aplysia juliana were also tested against two addition strains of Candida albicans (AMMRL36.42, AMMRL36.70). The largest zones of inhibition were consistently produced against strain AMMRL36.42. The zones of inhibition produced against AMMRL36.70 were similar to those produced against ACM4581.

The egg ribbons of Aplysia juliana were found to inhibit marine pathogens, in addition to the four human pathogens. Large zones of inhibition (>1cm) were produced against Vibrio anguillarium. Minimal (< 0.5 cm) but consistent zones of inhibition surrounded the egg ribbons on lawns of Vibrio harveyi and Vibrio alginolyticus. Chapter 4:Antimicrobial activity in molluscan egg masses 176

Table 4.4: The antimicrobial properties of a range of molluscan and polychaete egg masses, tested in the zone of inhibition assay on agar plates. The test microorganisms are Staphylococcus aureus ACM844 (S.a.), Escherichia coli ACM845 (E.c.), Pseudomonas aeruginosa ACM846 (P.a.) and Candida albicans ACM4581 (C.a.). The symbols used in the table are as follows; c indicates contamination from the growth of foreign microorganisms surrounding the tissue; ++ denotes a large zone of inhibition (> 1cm diameter); + denotes a small but consistent zone of inhibition (< 1cm); i denotes an inconsistent zone of inhibition around the tissue; - indicates no apparent zone of inhibition; NT indicates the sample was not tested against the particular microorganism.

Species Preparation Zone of Inhibition S.a. E.c. P.a. C.a. PHYLUM: MOLLUSCA, CLASS: GASTROPODA Nerita atramentosa Crushed eggs c c - - Bembicium nanum Crushed eggs c c - - Freeze-dried eggs - - - - Conuber cf. sordidus Crushed eggs c c c c Intact egg mass c c c c Dicathais orbita Crushed capsules + i - - Intact capsules - - - - Freeze-dried egg mass - - - - Intracapsular fluid + + - i Agnewia tritoniformis Crushed capsules ++ + + i Trunculariopsis trunculus Freeze-dried egg mass i - NT i Ceratostoma erinaceum Freeze-dried egg mass + i NT i Mitra carbonaria Crushed egg capsules + + - - Conus paperliferus Crushed egg capsules - + NT - Unidentified sp. 2 Crushed egg capsules i + - - Salinator fragilis Crushed egg ribbons c c - - Salinator solidus Crushed egg ribbons c c c c Siphonaria denticulata Crushed egg ribbons + + + + Intact egg ribbons + - - i Isidorella hainesi Crushed egg mass + - - - Glyptophysa gibbosa Crushed egg mass + i i - Meridolum gulosum Crushed egg mass + + NT NT Unidentified sp. 14 Crushed egg mass c c - - Aplysia juliana Crushed egg ribbons ++ ++ + + Intact egg ribbons + + - - Frozen egg ribbons ++ ++ NT + Freeze-dried egg ribbons + + NT + Aplysia sydneyensis Crushed egg ribbons ++ + ++ + Intact egg ribbons c - - - Aplysia parvula Crushed egg ribbons ++ ++ ++ ++ Aplysia dactylomela Crushed eggs ribbons ++ + ++ + Intact egg ribbons - - - NT Stylocheilus longicauda Crushed egg ribbons - - - - Dolabrifera dolabrifera Crushed egg ribbons + i + - Intact egg ribbons - - + - Philine angasi Crushed egg mass - i - -

Table 4.4 continued. Chapter 4:Antimicrobial activity in molluscan egg masses 177

Species Preparation Zone of Inhibition S.a. E.c. P.a. C.a. Pleurobranchus peroni Intact egg ribbons - - - - Pleurobranchea sp. Crushed egg ribbons i + NT - Bullina lineata Crushed egg ribbons c i NT - Hydatina physis Crushed egg ribbons c c - - Oxynoe viridis Crushed egg ribbons NT + NT NT Dendrodoris fumata Crushed egg ribbons + + NT - Platyodoris sp. Crushed egg ribbons + + NT - Jorunna pantherina Intact egg ribbons i i - - Goniodoris sp Intact egg ribbons - - - - Plocampherus imperialis Intact egg ribbons - - - - Spurilla neopolitana Freeze-dried eggs - - - - Unidentified sp. 13 Crushed eggs + + + NT

PHYLUM: MOLLUSCA, CLASS: CEPHALOPODA Sepioteuthis australis Freeze-dried egg mass - - NT -

PHYLUM: ANNELIDIA, CLASS: POLYCHAETA Eupolymnia sp. Freeze-dried egg mass + i NT - Unidentified sp. 1 Crushed egg mass - i i - Unidentified sp. 2 Crushed egg mass - i i NT Unidentified sp. 3 Crushed egg mass c c - -

Chapter 4:Antimicrobial activity in molluscan egg masses 178

Figure 4.2: White filamentous fungi surrounding the egg ribbons of the marine pulmonate Salinator fragilis. The egg ribbons have been placed on nutrient agar spread with Escherichia coli and tested for antimicrobial activity in the zone of inhibition assay.

Figure 4.3: The antimicrobial activity of the egg ribbons from Aplysia juliana tested against Staphylococcus aureus on nutrient agar. The samples on the left have been crushed with a mortar and pestle. The samples on the right have been placed intact onto the agar.

Chapter 4:Antimicrobial activity in molluscan egg masses 179

4.4.4 Fluorescein Diacetate assay

Cultures of Gram negative bacteria, E. coli and P. aeruginosa, as well as the four marine bacteria, were found to rapidly hydrolyse FDA producing clearly visible results within 3 hours. S. aureus typically took 3.5 - 4 hours before the production of FDA was clearly visible. There was no difference in FDA production between the bacterial cultures incubated with acetone or Milli Q water. On the other hand, very inconsistent results were obtained using

Candida albicans (ACM4581). On several occasions FDA hydrolysis could not be detected after a 24 hr incubation period. Two additional strains of C. albicans

(AMMRL 36.42, AMMRL 36.70) were tested with similar results (Appendix 4.2).

Organic extracts from the egg masses of 22 molluscs and one polychaete were found to inhibit the hydrolysis of FDA by at least one human pathogen (Table

4.2, 4.5). All of these species inhibited the growth of both Gram positive and

Gram negative bacteria, although not always at the same concentration (Table

4.5). No antimicrobial activity was observed with the seawater or solvent controls (Table 4.6). However, extracts taken from estuarine mud and intertidal pebbles did inhibit FDA hydrolysis at concentrations of 10mg/ml (Table 4.6).

The lipophilic extracts from most species only showed clear antimicrobial activity at a concentration of 10mg/ml (Table 4.5). Although the Gram positive bacteria Staphylococcus aureus was susceptible to some of the egg extracts at

1mg/ml. Antimicrobial activity was often observed at 1mg/ml in the non-polar fractions of the chloroform extracts. Strong antimicrobial activity was only detected in the polar extracts from three species in the family Aplysiidae and Chapter 4:Antimicrobial activity in molluscan egg masses 180

one fresh water pulmonate Isidorella hainesi (Table 4.5). The water/methanol extracts from the egg masses of Philine angasi, Salinator fragilis and an unidentified polychaete (sp. 3) partially inhibited the hydrolysis of FDA by the

Gram negative bacteria. The water/methanol extracts from the eggs of several species were tested at three to five times the concentration of the lipophilic extracts but still showed no significant antimicrobial activity (Table 4.5). It was not possible to determine the activity in the polar extract of Conus paperliferus egg capsules because broth controls containing this extract were found to hydrolyse FDA (Table 4.5). This was the only extract that was found to autofluoresce.

Extracts from the egg masses of three species (Conuber cf. sordidus, Dicathais orbita and Aplysia juliana) were prepared in a range of different solvents and these were found to inhibit the growth of the bacteria at different concentrations

(Table 4.5). Extracts using the least polar solvents, ether and chloroform, were the most effective for Dicathais orbita and Conuber cf. sordidus. The difference between the chloroform and ether extracts of Dicathais orbita egg capsules may be explained partly by the fact that the chloroform extract was less soluble in acetone than the ether extract. No activity was detected in the water/methanol layer from the chloroform/methanol extract and minimal activity was found in an ethanol extract (Table 4.5). Both the chloroform and water/methanol layers of the extract from Aplysia juliana egg ribbons showed greater activity against all three bacteria than the ethanol extract (Table 4.5).

The egg mass of Sepioteuthis australis was extracted and tested for antimicrobial activity on two separate occasions. The first extract was prepared Chapter 4:Antimicrobial activity in molluscan egg masses 181

in chloroform/methanol and this extract showed no activity. Notably, this extract frothed during the process of evaporation and produced a foamy liquid that appeared to decompose. On the second occasion, the egg mass was freeze- dried prior to extraction with chloroform. This extract did not foam or appear to decompose and showed mild activity against the two test bacteria (Table 4.5).

Extracts were taken from Dicathais orbita egg masses that were retained in the freezer for six months and these did not retain antimicrobial activity. However, the egg mass of Aplysia juliana retained antimicrobial activity after storage for one year at –200C. The fresh extracts from all of the other species showed consistent results when tested in different assays.

In general, the Gram positive bacterium Staphylococcus aureus was more susceptible to the antimicrobial properties in the egg masses than the two Gram negative bacteria. Staphylococcus aureus was completely inhibited by extracts from a greater number of species and more of the fractions showed some activity against this bacterium (Table 4.5). The Gram negative bacterium

Pseudomonas aeruginosa was slightly more resistant to the egg extracts than the other Gram negative bacterium Escherichia coli. The extracts and fractions from the egg masses of several species completely inhibited FDA hydrolysis by

E. coli but did not inhibit or only partially inhibited P. aeruginosa. Interestingly, different fractions or solvent extracts from the egg masses of five species

(Salinator fragilis, Spurilla neopolitana, Philine angasi, and the two polychaetes) showed differential activity against the Gram positive and Gram negative bacteria (Table 4.5). In each of these cases the Gram negative bacteria were inhibited by more polar fractions or extracts than the Gram positive bacterium.

Chapter 4:Antimicrobial activity in molluscan egg masses 182

Table 4.5: Bacteriostatic activity in the egg masses and adult tissue of a molluscs from two subclasses, as well as the egg masses from 2 polychaete worms, tested in the Fluorescein diacetate (FDA) hydrolysis assay. Chloroform and water/methanol (MeOH) extracts have been tested for each species, as well as three fractions of the chloroform extract. Fraction 1 is the most lipophilic and Fractions 2 and 3 are of increasingly polarity. The test microorganisms were Staphylococcus aureus ACM844 (S.a.), Escherichia coli ACM845 (E.c.) and Pseudomonas aeruginosa ACM846 (P.a.). The symbols used in the table are as follows; ++ indicates complete suppression of FDA hydrolysis; + denotes partial FDA hydrolysis but not at the level observed in the control cultures; – indicates no antimicrobial activity i.e. maximum FDA hydrolysis; AF denotes autofluorescence of the test sample in broth with FDA but no microbial cells; NT indicates the sample was not tested against the given bacteria. Species Sample Concentration Bacteriostatic activity mg/ml S.a. E.c. P.a. PHYLUM: MOLLUSCA, CLASS: GASTROPODA Bembicium nanum Chloroform 10 ++ ++ + Fraction 1 10 ++ - - Fraction 2 10 ++ ++ ++ Fraction 3 10 + + + MeOH/water 30 - - - Conuber c.f sordidus Chloroform 10 ++ ++ ++ Fraction 1 1 + + + Fraction 2 1 ++ ++ ++ Fraction 3 1 + + + MeOH/water 30 - + - Ether 10 ++ ++ NT Cabestana spengleri Chloroform 10 ++ ++ ++ Fraction 1 1 ++ ++ ++ Fraction 2 1 - - - Fraction 3 1 - - - MeOH/Water 10 - - - Dicathais orbita Chloroform/MeOH 10 ++ ++ + Chloroform 1 ++ + + 10 ++ ++ ++ Fraction 1 1 ++ ++ ++ Fraction 2 1 ++ ++ + Fraction 3 1 - - - MeOH/Water 50 - - - Ether 0.1 ++ ++ + 1 ++ ++ ++ Ethanol 10 ++ ++ + Agnewia tritoniformis Chloroform 10 ++ ++ + Fraction 1 1 + - - Fraction 2 1 ++ + +

Chapter 4:Antimicrobial activity in molluscan egg masses 183

Table 4.5 continued Species Sample Concentration Bacteriostatic activity mg/ml S.a. E.c. P.a. Agnewia tritoniformis continued. Fraction 3 3 + - - MeOH/water 10 - - - Bedeva hanleyi Chloroform 10 ++ ++ + MeOH/Water 10 - - - Lepsiella reticularis Chloroform 10 ++ ++ ++ MeOH/water 10 - - - Morula marginalba Chloroform 10 ++ ++ ++ MeOH/water 10 - - - Trunculariopsis trunculus Chloroform 1 ++ - NT 10 ++ + NT Fraction 1 1 + + NT Fraction 2 1 ++ ++ NT Fraction 3 1 ++ ++ NT Ceratostoma erinaceum Chloroform 1 ++ + NT 10 ++ ++ NT Fraction 1 1 ++ ++ NT Fraction 2 1 + ++ NT Fraction 3 1 - - NT Mitra carbonaria Chloroform 10 - - - Fraction 1 1 - - - Fraction 2 1 - - - Fraction 3 1 - - - MeOH/water 10 - - - Conus paperliferus Chloroform 1 + ++ NT 10 ++ ++ NT Fraction 1 1 ++ + NT Fraction 2 1 ++ ++ NT Fraction 3 1 + - NT MeOH/water 10 AF AF AF Salinator fragilis Chloroform 10 ++ ++ ++ Fraction 1 1 ++ ++ ++ Fraction 2 1 ++ ++ ++ MeOH/Water 10 - + + Salinator solidus Chloroform 5 ++ ++ ++ Fraction 1 3 + + + Fraction 2 3 + + + Fraction 3 3 ++ ++ ++

Chapter 4:Antimicrobial activity in molluscan egg masses 184

Table 4.5 continued. Species Sample Concentration Bacteriostatic activity mg/ml S.a. E.c. P.a. Salinator solidus continued. Fraction 4 25 + + + MeOH/Water 50 - - - Siphonaria denticulata Chloroform/MeOH 10 ++ - - Chloroform 10 ++ ++ + Fraction 1 1 ++ ++ ++ Fraction 2 1 ++ ++ ++ Fraction 3 20 - - - MeOH/water 10 - - - Siphonaria zelandica Chloroform 10 ++ ++ + Fraction 1 1 ++ ++ + Fraction 2 1 ++ ++ + Fraction 3 1 - - - MeOH/water 10 - - - Isidorella hainesi Chloroform 10 ++ ++ ++ Fraction 1 1 + + + Fraction 2 1 ++ ++ ++ MeOH/Water 10 ++ ++ ++ Aplysia juliana Chloroform/MeOH 1 ++ - - 10 ++ ++ ++ Chloroform 1 ++ + - MeOH/water 1 ++ ++ - 10 ++ ++ + Ethanol 10 ++ ++ NT Aplysia sydneyensis Chloroform 10 ++ + - MeOH/water 10 ++ ++ ++ Stylocheilus longicauda Chloroform 10 ++ ++ ++ Fraction 1 1 + + + Fraction 2 1 ++ ++ ++ Fraction 3 1 + + + MeOH/water 1 - - ++ 10 ++ ++ ++ Philine angasi Chloroform 10 ++ ++ - MeOH/water 50 - + + Spurilla neopolitana Chloroform 10 ++ - NT Fraction 1 1 - - NT Fraction 2 1 ++ - NT Fraction 3 1 - ++ -

Chapter 4:Antimicrobial activity in molluscan egg masses 185

Table 4.5 continued. Species Sample Concentration S.a. E.c. P.a. mg/ml PHYLUM: MOLLUSCA, CLASS: CEPHALOPODA Sepioteuthis australis Chloroform/MeOH 10 - - - Chloroform 10 ++ + NT Fraction 1 1 ++ - NT Fraction 2 1 ++ ++ NT Fraction 3 1 + + NT PHYLUM: ANNELIDIA, CLASS: POLYCHAETA Eupolymnia sp. Chloroform 10 ++ - NT Fraction 1 1 ++ - NT Fraction 2 1 ++ - NT Fraction 3 1 + + NT Unidentified sp. 3 Chloroform 10 + - - MeOH/water 10 - + +

Table 4.6: Bacteriostatic activity of a number of control samples tested in the Fluorescein diacetate (FDA) hydrolysis assay. The test microorganisms are Staphylococcus aureus (S.a.), Escherichia coli (E.c.) and Pseudomonas aeruginosa (P.a.). The symbols used in the table are; ++ complete inhibition of FDA hydrolysis; + partial inhibition of FDA hydrolysis; - no detectable inhibition of FDA hydrolysis.

Sample Solvent Concentration Bacteriostatic activity mg/ml S.a. E.c. P.a. Sea water Chloroform 10 - - - Estuarine mud Chloroform/ MeOH 10 ++ ++ ++ Intertidal pebbles Chloroform 10 ++ ++ ++ Dried solvent Chloroform 10 - - - Dried solvent MeOH/water 10 - - - Dried solvent Ether 10 - - - Dried solvent Ethanol 10 + + - Dried solvent Ethanol 1 - - -

Chapter 4:Antimicrobial activity in molluscan egg masses 186

The extracts from the egg masses of Dicathais orbita and Aplysia juliana were tested for antimicrobial activity against a number of marine pathogens and a mixed culture of skin flora (Table 4.7). The chloroform extracts from D. orbita extracts completely inhibited the FDA hydrolysis by all of the marine pathogens and the skin flora at a concentration of 10mg/ml. The extracts also partially inhibited the growth of three Vibrio spp. at 1mg/ml. The chloroform extracts of

Aplysia juliana completely inhibited the growth of Vibrio anguillarium at 10mg/ml but only partially inhibited Enterococcus sericolicida and the skin flora at this concentration. Water/methanol extracts from Aplysia juliana egg masses only partially inhibited the marine bacteria and skin flora at concentrations of

100mg/ml. These polar extracts were not desalted prior to use in the FDA assay.

Table 4.7: Antimicrobial activity in the extracts from the egg masses of Dicathais orbita and Aplysia juliana tested against four marine pathogens and a mixed culture of skin flora (S.F.) in the Fluorescein diacetate (FDA) hydrolysis assay. The marine microorganisms are Vibrio anguillarium (V.an.), Vibrio harveyi (V.h.), Vibrio alginolyticus (V.al.); Enterococcus sericolicida (E.c.). The symbols used in the table are; ++ complete inhibition of FDA hydrolysis; + partial inhibition of FDA hydrolysis; - no detectable inhibition of FDA hydrolysis; NT indicates the sample was not tested against the particular microorganism.

Species Sample Concentration Bacteriostatic activity mg/ml V.an. V.h. V.al. E.s. S.F. Dicathais orbita Chloroform 1 + + + - - 10 ++ ++ ++ ++ ++ Aplysia juliana Chloroform 1 - NT NT - - 10 ++ NT NT + + MeOH/water 10 - NT NT - - 100 + NT NT + +

Chapter 4: Antimicrobial activity in molluscan egg masses 187

4.4.6: Antimicrobial (cell stasis/lysis) activity

Plating out the cultures from the FDA assay onto fresh agar only permits the detection of a high rate of cell death. By comparison with a dilution series of the control cultures, it was possible to detect a 90% reduction in the cell population for Staphylococcus aureus, Escherichia coli and Candida albicans. However, it was only possible to detect a reduction of greater than 99% of the population for the faster growing Pseudomonas aeruginosa and marine bacteria. The results presented in Table 4.8 were not always repeatable in separate assays and this was probably because of the high rate of cell death required to detect activity.

Extracts from the egg masses of all of the species of molluscs tested in the cell lysis assay were at least partially lethal to the Gram positive bacterium

Staphylococcus aureus (Table 4.8). On the other hand only a small number of extracts or fractions were bactericidal against the Gram negative bacteria

(Table 4.8). However, extracts from the egg masses of Spurilla neopolitana appear to have greater bactericidal activity against the Gram positive bacterium

Escherichia coli than S. aureus. This is consistent with the results of the FDA assay (Table 4.5).

The chloroform extract from Conuber cf. sordidus killed at least 90% of the population of Pseudomonas aeruginosa. This bactericidal activity appears to be restricted to Fraction 2 of the extract. Fraction 2 from Conuber cf. sordidus also killed 99% of the S. aureus population and at least 90% of the populations of

Escherichia coli and Candida albicans. This was the most widespread activity that was observed. Fraction 1 of the chloroform extract from Bembicium nanum Chapter 4: Antimicrobial activity in molluscan egg masses 188

also showed some bactericidal activity against P. aeruginosa and C. albicans.

Fraction 3 of the chloroform extract from Salinator solidus was strongly bactericidal to both Escherichia coli and S. aureus but not P. aeruginosa or C. albicans. Escherichia coli was also susceptible to the chloroform extract from

Aplysia juliana but only at very high concentrations (i.e. 50mg/ml). Candida albicans was inhibited by fractions or extracts from seven out of the ten species tested (Table 4.8).

The extracts from the egg masses Dicathais orbita and Aplysia juliana were tested against two additional strains of Candida albicans (AMMRL 36.42,

AMMRL 36.70). One of these strains (AMMRL 36.70) was more susceptible to the antimicrobial compounds in these egg masses, with over 99% of the population killed by the chloroform extracts. Antimicrobial activity against the other strain (AMMRL36.42) was similar to that summarised in Table 4.8 for strain ACM 4581.

The egg masses of Dicathais orbita and Aplysia juliana were also tested for bactericidal activity against a number of marine pathogens and a mixed culture of skin flora (Table 4.9). Chloroform extracts from D. orbita egg masses were found to be highly bactericidal to three marine Vibrio spp., as well as the mixed skin flora. This extract was also bactericidal against Enterococcus sericolicida causing approximately 90% cell death at a concentration of 10mg/ml. However, it should be noted that the control cultures of Enterococcus sericolicida were only just visible on the agar surface. Therefore the results for this marine bacterium are subjective. Chapter 4: Antimicrobial activity in molluscan egg masses 189

Table 4.8: Antimicrobial (cytolytic) activity in the egg masses of 11 marine molluscs and one polychaete determined by assessing the recovery of microbial cultures after being incubated with extracts from the egg masses and then spread onto fresh agar medium. Chloroform and water/methanol (MeOH) extracts have been tested, as well as three fractions of the chloroform extract. Fraction 1 was the most lipophilic, with fractions 2 and 3 increasingly in polarity respectively. The test microorganisms are Staphylococcus aureus (S.a.), Escherichia coli (E.c.), Pseudomonas aeruginosa (P.a.) and Candida albicans (C.a.). The symbols used in the table are as follows; ++ denotes less than 1% viability; + denotes 1-10% viability; - indicates no detectable activity; NT indicates the sample was not tested.

Species Sample Concentration Cytolytic activity mg/ml S.c. E.c. P.a. C.a. PHYLUM: MOLLUSCA, CLASS: GASTROPODA Bembicium nanum Chloroform 10 + - - - Fraction 1 1 ++ - + + Fraction 2 1 + - - - Fraction 3 1 - - - - MeOH/ water 30 - - - - Conuber cf. sordidus Chloroform 10 ++ - + + Fraction 1 1 + - - + Fraction 2 1 ++ + + + Fraction 3 1 - - - + MeOH/ water 30 - - - - Dicathais orbita Chloroform 10 + - - ++ Fraction 1 1 ++ + - NT Fraction 2 1 + - - NT MeOH/ water 10 - - - - Trunculariopsis trunculus Chloroform 10 + - NT NT Fraction 1 1 + - NT NT Fraction 2 1 - - NT NT Fraction 3 1 - - NT NT Ceratostoma erinaceum Chloroform 10 + + NT NT Fraction 1 1 + + NT NT Fraction 2 1 + - NT NT Fraction 3 1 - - NT NT Conus paperliferus Chloroform 10 + - NT NT Fraction 1 1 + + NT NT Fraction 2 1 + - NT NT Fraction 3 1 + - NT NT Water/ MeOH 10 - - NT NT Salinator fragilis Chloroform 10 ++ - - +

Chapter 4: Antimicrobial activity in molluscan egg masses 190

Table 4.8 continued.

Species Sample Concentration Cytolytic activity mg/ml S.c. E.c. P.a. C.a. Salinator fragilis continued. Fraction 1 1 ++ - - ++ Fraction 2 1 ++ - - ++ Salinator solidus Chloroform 10 + - - - Fraction 1 1 - - - - Fraction 2 1 - - - - Fraction 3 1 ++ ++ - - MeOH/ water 50 - - - - Siphonaria denticulata Chloroform 10 ++ - - - Fraction 1 1 ++ - - - Fraction 2 1 ++ - - - Fraction 3 1 - - - - MeOH/ water 10 - - - - Siphonaria zelandica Chloroform 10 ++ - - NT Fraction 1 1 ++ - - NT Fraction 2 1 ++ - - NT Isidorella hainesi Chloroform 10 ++ - - ++ Fraction 1 1 - - - ++ Fraction 2 1 ++ - - ++ MeOH/ water 10 + - - - Aplysia juliana Chloroform 50 ++ ++ - NT 10 ++ - - ++ MeOH/ water 10 ++ - - - Stylocheilus longicauda Chloroform 10 ++ - - NT Fraction 1 1 ++ - - NT Fraction 2 1 ++ - - NT Fraction 3 1 - - - NT MeOH/ water 10 ++ - - NT Philine angasi Chloroform 10 + - - + MeOH/ water 10 - - - - Spurilla neopolitana Chloroform 10 - + NT NT Fraction 1 1 - - NT NT Fraction 2 1 + + NT NT Fraction 3 1 - + NT NT PHYLUM: MOLLUSCA, CLASS: CEPHALOPODA Sepioteuthis australis Chloroform 10 ++ - NT NT Fraction 1 1 - - NT NT Fraction 2 1 + - NT NT Fraction 3 1 ++ - NT NT

Chapter 4: Antimicrobial activity in molluscan egg masses 191

Table 4.8 continued.

Species Sample Concentration Bactericidal activity mg/ml S.c. E.c. P.a. C.a. PHYLUM: ANNELIDIA, CLASS: POLYCHAETA Eupolymnia sp. Chloroform 10 + - NT NT Fraction 1 1 + - NT NT Fraction 2 1 + - NT NT Fraction 3 1 + - NT NT Unidentified sp.1 Chloroform 10 ++ - - - MeOH/ water 10 - - - -

Table 4.9: Bactericidal activity in the extracts from the egg masses of Dicathais orbita and Aplysia juliana tested against four marine pathogens and a mixed culture of skin flora (S.F.). Bactericidal activity was determined by assessing the recovery of microbial cultures after being incubated with extracts and then spread onto fresh agar medium. The marine microorganisms are Vibrio anguillarium (V.an.), Vibrio harveyi (V.h.), Vibrio alginolyticus (V.al.); Enterococcus sericolicida (E.c.). The following symbols are used; ++ denotes less than 1% viability; + denotes 1-10% viability; - indicates no detectable activity; NT indicates the sample was not tested.

Species Sample Concentration Bactericidal activity mg/ml V.an. V.h. V.al. E.s. S.F. Dicathais orbita Chloroform 1 + + + NT + 10 ++ ++ ++ + ++ Aplysia juliana Chloroform 1 - NT NT - - 10 - NT NT - + MeOH/Water 10 - NT NT - -

Chapter 4: Antimicrobial activity in molluscan egg masses 192

Bactericidal activity against the marine bacteria was not detectable in either the polar or lipophilic extracts from the eggs of Aplysia juliana (Table 5.9). The chloroform extract from A. juliana caused approximately 90% cell death of the mixed skin culture at fairly high concentrations (50mg/ml).

4.4.7 Epibiosis and microfouling

All of the molluscan egg masses that were placed on marine agar and incubated overnight at 250C produced white or cream colonies in the region surrounding the egg masses (Table 4.10; Appendix 4.3). These colonies were initially localised around the eggs but spread across the plate after a few days of incubation. Similar bacterial colonies were observed around a proportion of the egg masses that were placed on nutrient agar and tested for antimicrobial activity in the zone of inhibition assay (Table 4.4; 4.10; Appendix 4.3). A white filamentous fungus was also observed around the egg masses of some species that were placed on nutrient agar (Table 4.10; Appendix 4.3).

The cream or white bacterial colonies were also observed around samples of intertidal sand and estuarine mud on marine agar. The white filamentous fungus was observed on nutrient agar, surrounding samples of estuarine mud.

Significantly, four out of five of the egg masses that produced the white filamentous fungus were derived from estuarine species (Appendix 4.3). Further details on epibiotic organisms are provided in Appendix 4.3.

Chapter 4: Antimicrobial activity in molluscan egg masses 193

Table 4.10: The proportion of invertebrate egg masses found with associated microfauna after an overnight incubation on Marine Agar at 250C or Nutrient Agar at 370C.

Substrate Epibiotic organism Proportion of species Marine agar Cream or white bacterial colonies 12/12 Nutrient agar Cream or white bacterial colonies 12/41 Nutrient Agar White filamentous fungi 5/41

4.5 Discussion

4.5.1 Antibiotics in invertebrate egg masses

This study provides evidence for the evolution of antimicrobial protection in the egg masses of marine invertebrates. The egg masses from 46 invertebrates were tested for antimicrobial activity against a range of human pathogens and some activity was found in 78% of these species (Table 4.2).

The egg masses from Dicathais orbita and Aplysia juliana were also found to inhibit the growth of four marine pathogens (Table 4.), three of which are known to infect molluscs (refer to Austin, 1988; Sutton and Garrick, 1993;

Koh, 1997). This suggests that the antimicrobial components in these egg masses play an ecological role, in the prevention of microbial infection. The idea that egg masses of marine invertebrates provide protection against microbial infection was first suggested by Thorson (1950) but until now has not been rigorously tested.

Antibiotic properties appear to be widespread in the egg masses of marine invertebrates. Antimicrobial activity was observed in the egg masses of molluscs from a broad range of families, as well as two polychaetes (Table

4.2). The two major forms of molluscan egg mass (gelatinous egg masses

Chapter 4: Antimicrobial activity in molluscan egg masses 194 and leathery egg capsules) were both found to have antimicrobial activity. The requirement for antimicrobial components in the egg masses of molluscs also appears to extend into the terrestrial environment. The egg masses from two fresh water snails and one land snail were found to inhibit microbial growth

(Table 4.2). These findings support the idea that invertebrates egg masses are vulnerable to microbial infection and require chemical protection. This is consistent with the high incidence of chemical defence that has been found associated with the reproductive tissue of some other terrestrial (e.g. Blum et al., 1959; Wilson, 1971; Beattie et al., 1986; Hare and Eisner, 1993) and marine invertebrates (e.g. Hart et al., 1979; Harvell, 1984; Coll et al., 1985;

Gil-Turnes et al., 1989; Gil-Turnes and Fenical, 1992; McClintock and Vernon,

1990).

Antimicrobial protection of benthic egg masses appears to have evolved at least twice within the Mollusca. The deposition of benthic egg masses has evolved in two separate classes of mollusc, Gastropoda and Cephalopoda

(Smith et al., 1989) and species from both of these classes were found to have antimicrobial activity in their egg masses (Table 4.2). Within the class

Gastropoda, egg masses have evolved in one subclass, the Orthogastropoda, but are thought to have evolved independently in two clades; the apogastropods (incl. caenogastropods and heterobranchs) and the neritopsines (incl. Neritoidea) (Ponder and Linberg, 1997). Most of the species tested in this study are apogastropods. The egg capsules from one species of Neritoidea (Nerita atramentosa) was tested from antimicrobial activity in the zone of inhibition assay but did not show clear antimicrobial

Chapter 4: Antimicrobial activity in molluscan egg masses 195 activity due to contamination by foreign microorganisms surrounding the eggs

(Table 4.4). Future research should include testing the egg masses from this species in the Fluorescein diacetate assay, as well as testing a greater range of species from the gastropod superorder Neritopsina and the class

Cephalopoda.

Variation in the antimicrobial components found in molluscan egg masses could exist, not only between species, but also between populations of the same species. There was no detectable variation in the level of antimicrobial activity of replicate samples from the same species tested in the zone of inhibition and FDA assays. However, some intraspecies variation was found in the bactericidal activity of extracts taken from molluscan egg masses that were collected on different headlands in the Wollongong area. This is most likely a result of the limitations of the assay used to detect bactericidal activity.

Only a very high rate of cell death (>90%) could be detected using this assay and therefore minor variations in the growth rate of the different cultures could produce different results. Nevertheless, variation in the biological activity of marine organisms has previously been found between intertidal and subtidal populations of sponge (Thompson, 1985) and from geographically isolated populations of coral (Harvell and Fenical, 1989) and algae (Targett et al.,

1992). Different metabolites have also been isolated from ascidians collected at different locations (Rinehart et al., 1987; Li and Blackman, 1995).

Ultimately, this type of intraspecies variation could increase the overall resource potential of marine biological diversity.

Chapter 4: Antimicrobial activity in molluscan egg masses 196

It is also possible that there is some variation in the antimicrobial properties between different individuals within a species. This is particularly likely if the antimicrobial compounds are diet derived, as appears to be the case for the biologically active compounds that have been found in the egg masses of the

Opisthobranchs Hexabranchus sanguineus (Faulkner, 1992a) and Elysia halimedae (Paul and van Alstyne, 1988). Significantly, all of the molluscs that did not show clear antimicrobial activity in this study, were only tested using the egg mass from one or two individuals. Furthermore, the egg masses from several species were collected from aquaria (Table 4.1). The deposition of egg masses by Opisthobranchs in aquaria is thought to be a stress response

(Bill Rudman, pers. com.) and therefore these species may not have accumulated the compounds that are normally produced to protect the egg mass. Significantly, soft corals have been shown to accumulate the defensive compounds found in the eggs only a few weeks prior to spawning (Coll et al.,

1985). Consequently, it remains possible that antimicrobial activity in molluscan egg masses is even more widespread than indicated by this study.

4.5.2 The resource potential of molluscan antibiotics

The egg masses of marine molluscs have shown significant antimicrobial activity against a suite of human pathogens of considerable medical importance. In general, the Gram positive bacterium Staphylococcus aureus was more susceptible to the toxic compounds in the eggs than the other test organisms (Table 4.4; 4.5; 4.8). Staphylococcus aureus is an important human pathogen and has evolved resistance to a large number of conventional antibiotics in the last decade (Cohen, 1992, Neu, 1992, Cannon,

Chapter 4: Antimicrobial activity in molluscan egg masses 197

1995; Stinson, 1996). In contrast, the egg masses from only a few species of mollusc demonstrated bactericidal activity against the Gram negative bacteria

(Table 4.8). The greater resistance of the Gram negative bacteria to the molluscan egg masses in comparison to S. aureus may be due to the greater reinforcement in their cell walls (e.g. Martin and Beveridge, 1986). The eukaryotic microorganism Candida albicans was also inhibited by fewer egg masses than S. aureus. Lack of antimicrobial activity against eukaryotic cells could be an advantageous feature for the development of human or agricultural antibiotics.

The discovery of antibiotics that are effective against marine pathogens is of potential interest for the aquaculture industry where disease has become a considerable problem (Westra, 1998). The egg masses from two molluscs

(Dicathais orbita and Aplysia juliana) effectively inhibited the growth of several marine pathogens that can infect economically important fish stocks (refer to

Austin, 1988; Sutton and Garrick, 1993; Koh, 1997). However, extreme caution should be taken in the development of marine natural products for wide spread use in aquaculture. The potential evolution of resistance towards the natural product could create serious problems for the source organism and therefore these should only be considered as drug leads rather than for direct application.

Both broad-spectrum and specific antibiotics could be found in the egg masses of marine molluscs. For example, the egg masses of Dicathais orbita exhibited similar antimicrobial activity against a range of human pathogens,

Chapter 4: Antimicrobial activity in molluscan egg masses 198 marine pathogens and a mixed culture of skin flora (Table 4.4, 4.5, 4.7, 4.8).

These observations suggest that some molluscan egg masses contain components with broad-spectrum antimicrobial activity. Conversely, the extracts and fractions from some species differentially inhibited the different human pathogens (Table 4.5, 4.8). This is consistent with the findings of

Rinehart et al., (1981) where the extracts of different marine invertebrates were found to be more active against certain microorganisms. It is therefore possible that some marine molluscs contain compounds with specific antimicrobial activity in their egg masses. Both broad-spectrum and specific antimicrobial agents could provide useful leads for drug development.

However, specifically targeted antimicrobial agents are less likely to lead to the rapid evolution of resistance than broad-spectrum antimicrobial agents

(Cohen, 1992).

It is possible that many of the species tested in this study will also display significant biological activity in different types of pharmaceutical assays. A number of molluscs have been shown to produce compounds with antineoplastic activity (e.g. Rinehart et al., 1981; Yamazaki, 1993; Yamazaki et al., 1984; 1985; 1990). Rinehart et al., (1981) found no molluscs with antiviral activity against Herpes simplex. However, there is a high abundance of viruses in the marine environment (Bergh et al., 1989), therefore the potential that molluscan egg masses are chemically defended against viruses should not be over looked. Ultimately, the lack of observed activity in one assay should not be used to rule out the potential of significant biological activity in a different pharmacological assay.

Chapter 4: Antimicrobial activity in molluscan egg masses 199

4.5.3 Developmental changes in the antimicrobial properties

This study provides evidence for a chemical ripening process in the egg masses of marine molluscs. The assessment of antimicrobial activity in the egg masses of seven molluscs, at different stages of development, has clearly indicated that the egg masses have significantly greater antimicrobial activity when first laid and then lose activity as the larvae develop and hatch (Table

4.3; Figure 4.1). The loss of antimicrobial activity during larval development has been observed previously in the egg mass of the sea hare Aplysia juliana

(Kamiya et al., 1988). Many plants also produce toxic compounds in immature fruits that ripen as the seed develops (Orians and Janzen, 1974). However, similar mechanisms of protecting embryos in a ripening toxic matrix have not been described in animals, with the exception of Aplysia juliana.

The loss of antimicrobial activity during larval development could be an important component of the hatching process in molluscan egg masses. The gelatinous egg masses from many species of mollusc are typically a lot less firm during the later stages of development and decompose soon after hatching (pers. obs.). Microbial degradation of the jelly matrix around the time of hatching may facilitate the release of the larvae or juveniles into the water column (Harris, 1975; Eyster, 1986). Chemical changes in the egg masses, such as the decomposition of antimicrobial compounds, could also play a role in triggering the hatching of the larvae. Further studies on the chemical changes in molluscan egg masses are required to determine the importance of these changes to the hatching process.

Chapter 4: Antimicrobial activity in molluscan egg masses 200

Chemical protection against microbial infection appears to be more common at the egg stage of marine molluscs than in the fully developed adults.

Antimicrobial activity was only detected in the adult tissue of one out of six species of molluscs that were tested in this study (Table 4.3). This species

(Spurilla neopolitana) is a soft-bodied nudibranch that may have similar requirements for defensive compounds at the reproductive and adult life stages. It is also significant that this species was breeding at the time of collection (Dr A. Davis, pers. com.). All of the other species that were tested at the adult stage were shelled molluscs, although the extracts of only one shelled species (Dicathais orbita) were tested in the Fluorescein Diacetate assay. Nevertheless, it is possible that the physical protection afforded by the shell overcomes the need for defensive compounds, as suggested previously by Faulkner (1992a). With regard to this, it is of interest that the pre-shelled embryos of Nucella lapillus suffer high mortality after premature removal from the egg capsules, whereas the shelled embryos survive, even in the absence of antibiotics (Pechenik et al., 1984). Furthermore, the well-developed egg masses containing the shelled veligers in this study did not show any antimicrobial activity, whereas the fresh eggs did (Table 4.3). These data provide further evidence that the development of a shell replaces the need for antimicrobial compounds.

4.5.4 Localisation of the antimicrobial components and autotoxicity

The antimicrobial components in the egg masses of many molluscs appear to be restricted to the internal matrix of the egg mass rather than on the surface.

Chapter 4: Antimicrobial activity in molluscan egg masses 201

The crushed egg masses from several species were found to produce a much larger zone of inhibition than the uncrushed egg masses (Table 4.4; Figure

4.3). Presumably, crushing the egg masses breaks up the external matrix to release the active constituents. The empty egg cases of Dicathais orbita and

Mitra carbonaria also did not inhibit the growth of any of the test organisms, which further confirms that the activity in these eggs is not the result of surface components. Consequently, symbiotic microorganisms on the surface are unlikely be responsible for the antimicrobial activity in many molluscan egg masses, as has been found for the eggs of some crustaceans (Gil-Turnes and Fenical, 1989; Gil-Turnes et al., 1989).

It has been suggested that it would be advantageous to produce toxic or repellent compounds on the outer surface of eggs, to isolate the toxic compounds from the developing embryos (Orians and Janzens, 1974). The incorporation of antimicrobial components into the internal matrix of molluscan egg masses leads to the question of how the developing embryos are protected from autotoxicity. Interestingly, the embryos of marine molluscs are contained in turgid envelopes within the gelatinous egg mass or leathery capsules (Eyster, 1986; Hickman, 1992). The permeability of these envelopes to most organic molecules is not known. However, preliminary observations have demonstrated that the envelopes are not permeable to salts, fixatives and embedding media (Eyster, 1986). Consequently, these envelopes could provide a means by which the developing embryos are separated from the toxic components in the egg masses. Nevertheless, the young larvae or veliger would still have to cross the toxic matrix in the egg mass upon

Chapter 4: Antimicrobial activity in molluscan egg masses 202 hatching. Consequently, the observed loss of antimicrobial activity during embryonic development may also be important, to protect the larvae from autotoxicity as they emerge from their internal envelopes.

Autotoxicity may be less of a problem if the same toxins that are produced to protect the eggs are also found in the adults. In this case both the adults and larvae could be resistant to the toxins (Orians and Janzen, 1974). For example, the toad, Bufo spp. produce a highly toxic compound to protect the eggs and tadpoles, that is also present in the adults (Voris and Bacon, 1966).

Similarly, the same toxic compounds have been found in the eggs, larvae and adults of the starfish Acathaster planci (Lucas et al., 1979) and the puffer fish

Fugu rubripes (Fuhrman et al., 1969). All of these species are presumed to be resistant to their own toxins. Nevertheless, in many species natural products are encapsulated within specialised glands or cells. For example, the trichomes of terrestrial plants, physodes of algae and spherolous cells of sponges are used to contain toxic compounds (Steinburg, pers. com.).

Consequently, autotoxicity appears to be a major problem for the embryos, juveniles and adults of many species.

It is likely that the adults are the source of the antibiotic agents found in the molluscan egg masses, despite the fact that most of the adult tissue tested in this study did not show antimicrobial activity. An egg generally has a finite resource base and therefore it is unlikely that the embryos would allocate resources towards the synthesis of defensive compounds (Orians and

Janzen, 1974). It is possible that the toxic compounds are concentrated in the

Chapter 4: Antimicrobial activity in molluscan egg masses 203 egg masses by the adults, as has been previously reported for the nudibranch

Hexabranchus sanguineus (Roesener and Scheuer, 1986) and an Australian soft coral (Coll et al., 1985). Alternatively, the antimicrobial compounds could be held in a non-toxic state by the adult and then enzymatically activated in the reproductive tract or egg mass. Interestingly, the egg masses of the sea hares Aplysia kurodai and Aplysia juliana were found to contain different antitumor glycoproteins to those found in the reproductive tract of the adults

(Kisugi et al., 1987; Kamiya et al., 1986; Kamiya et al., 1988). The glycoproteins found in the albumen glands of the adults are thought to be precursors of those found in the egg mass (Yamazaki, et al., 1990). It is also of interest that the egg masses of Hypselodoris infucata do not contain the defensive compounds of the adult nudibranch but rather a different terpenoid aldehyde (Karuso, 1987).

4.5.5 Properties of the antimicrobial components

The antimicrobial activity in the egg masses of many marine molluscs appears to be attributable to components of low to moderate polarity. The activity in the egg masses of the molluscs tested in this study was primarily restricted to the lipophilic extracts and their fractions in the Fluorescein diacetate assay

(Table 4.5). In general, one or two of the fractions inhibited microbial growth at a concentration that was ten times lower than the crude extract (Table 4.5), indicative of the concentration of an active component.

It is likely that the egg masses from some species contain several active components. In particular, both the chloroform solubles and water/methanol

Chapter 4: Antimicrobial activity in molluscan egg masses 204 soluble components from extracts of four species showed clear antimicrobial activity (Table 4.5). Three of these species are from the family Aplysiidae and both lipophilic and hydrophilic biologically active factors have been previously extracted from the Aplysiidae (sea hares). For example, cytolytic glycoproteins have been isolated from eggs and adult sea hares in saline solutions (Yamazaki et al., 1984; Kisugi et al., 1987). In addition, an ichthyotoxic acetogenin (Miyamoto, 1995) and cytotoxic terpenes (Hollenbeak et al., 1979; Kusumi et al., 1987) have been isolated from chloroform extracts of adult sea hares.

The polar extract from the egg masses of Conus paperliferus was found to hydrolyse fluorescein diacetate in the absence of bacterial cells (Table 4.5).

This suggests that the egg mass from this species contains some type of esterase. A large number of neurotoxic peptides have been isolated from the genus Conus (Olivera et al., 1990; Myers et al., 1993). These biologically active peptides would be hydrophilic but it is not known if they could be responsible for the observed hydrolysis of fluorescein diacetate. Conus paperliferius was the only species that produced an extract which autofluoresced in this study.

The antimicrobial components in the fresh egg capsules of Mitra carbonaria appear to react rapidly in air to produce an insoluble and inactive product. The fresh egg mass from this species is white but changes to a deep magenta

(black) in less than 10 minutes after the egg capsules are disrupted (pers. obs.). The intracapsular fluid from egg capsules that had turned black was

Chapter 4: Antimicrobial activity in molluscan egg masses 205 ineffective in inhibiting the growth of bacteria (Table 4.5). Similarly, extracts taken from the egg mass of Mitra carbonaria rapidly turned black and showed no antimicrobial activity in the FDA assay (Table 4.6). These data indicate that the active components from the fresh egg mass decompose in oxygen.

The stability of the antimicrobial compounds in molluscan egg masses appears to vary between species. The egg mass of Aplysia juliana was found to retain antimicrobial activity after one year of storage in the freezer, suggesting that the active components in these eggs are relatively stable. On the other hand the egg mass of Dicathais orbita did not retain activity after prolonged storage in the freezer. The loss of bioactivity during storage has been previously observed in marine samples (Rinehart, 1988) and consequently, biological screening should be carried out immediately after sample collection to maximise the chances of obtaining a positive result.

4.5.6 Screening methods for detecting antimicrobial activity

The Zone of Inhibition (ZI) assay and the Fluorescein Diacetate (FDA) assay are complementary methods for the preliminary detection antimicrobial activity in natural samples. Both assays have small sample requirements and are quick and easy to use. Furthermore, the results from the same species tested in replicate assays were reproducible for both of these assays. The low costs involved in setting up these assays means they can be useful for researchers working independently from well-established pharmaceutical research institutes. The FDA assay is also appropriate for bioassay guided fractionation of the active components (Chand et al., 1995). Isolated compounds can then

Chapter 4: Antimicrobial activity in molluscan egg masses 206 be sent for testing in a variety of specific enzyme inhibition and receptor antagonist bioassays.

The Zone of Inhibition assay used in this study can be useful for rapidly assessing antimicrobial activity in natural samples with minimal preparation.

This means that the assay can be useful for screening samples that rapidly decompose, such as the egg masses of Mitra carbonaria. Samples of egg material less than 1cm in length were found to have antimicrobial activity using the zone of inhibition assay. Consequently, this assay may be particularly valuable for detecting activity in small samples or samples from rare species, such as the egg ribbons of many nudibranchs.

All of the egg capsules that produced a clear zone of inhibition showed greater activity when crushed prior to being placed on the bacterial lawn (e.g.

Figure 4.3.). This indicates that crushing the material helps release the active components. The egg ribbons of three species of Opisthobranchs that did not produce zones of inhibition were not crushed prior to use in the assay (Table

4.4). This may explain the absence of detectable activity in these egg masses.

In future experiments samples should be crushed for use in the zone of inhibition assay. However, this should be done immediately prior to use, in order to prevent dehydration and other alterations to the components of the samples. Comparison of activity in crushed and intact samples can also provide some information about the distribution of the active components in the sample.

Chapter 4: Antimicrobial activity in molluscan egg masses 207

The detection of antimicrobial activity using the zone of inhibition assay can be complicated by contamination from the specimen (e.g. Figure 4.2). This problem could be overcome by freeze-drying the samples, although the mobility of the active components on agar may be reduced in the dried specimens (refer to Table 4.4). Freeze-frying could also result in the loss of some volatile active components.

The egg masses from a number of molluscs were found to be active in the

FDA assay but not the Zone of Inhibition assay (Table 4.2). Significantly, only the lipophilic extracts from these species tested positive in the FDA assay.

Therefore, it appears that highly lipophilic extracts do not migrate across the watery agar matrix in the Zone of Inhibition assay. The egg ribbons from

Aplysia spp. produced the largest zones of inhibition and polar extracts from these egg ribbons were found to have activity in the FDA assay (Table 4.5).

The lack of observed antimicrobial activity in the egg capsules of Nucella

(Thais) lapillus by Pechenik et al., (1984) may be due the lipophilicity or insolubility of the active compounds. These authors tested for antimicrobial activity in the intracapsular fluid of this mollusc using the disk diffusion assay.

In the current study, extracts from the egg capsules of seven species in the same family as N. lapillus (the Muricidae) were found to have activity in the

FDA assay (Table 4.2, 4.6). However, zones of inhibition were small and inconsistent around these egg masses (Tables 4.2, 4.3) emphasising that a failure to detect a zone of inhibition may not rule out the presence of antimicrobial compounds in the egg mass of N. lapillus.

Chapter 4: Antimicrobial activity in molluscan egg masses 208

The lack of observed antimicrobial activity in a lipophilic extract of the egg mass of Dendrodoris nigra by Matsunaga et al. (1986), may also have been due to the physical properties of the active compounds. In the present study, antimicrobial activity was observed in the crushed egg mass of D. nigra using the zone of inhibition assay. Consequently, the active components in the egg mass of this species could be hydrophilic.

The FDA assay proved very useful for rapidly screening for antimicrobial activity in crude extracts and semi-purified fractions. This assay requires only

40mg of extract to test three replicates of the sample against 4 different pathogens at a maximum concentration of 10mg/ml, with appropriate controls.

In general, 40mg of extract could be obtained from less than 20g of egg material. A high rate of antimicrobial activity was detected in egg masses of marine molluscs using this assay, with the extract from only one species testing negative (Table 4.5). However, one extract was found to autofluoresce in the presence of FDA, which demonstrates the need for appropriate controls.

Some problems were found with the growth of Candida albicans in the FDA assay. This microbe appears to be sensitive to the presence of acetone in the microtitre plate. However, acetone did not affect the growth of any of the bacteria used in this study, including marine and human pathogens. The Zone of Inhibition assay was found to be useful for C. albicans, as well as most of the bacteria. However, one marine bacterium Enterococcus sericolicida,

Chapter 4: Antimicrobial activity in molluscan egg masses 209 produced a very light lawn on the agar surface, making it difficult to distinguish a zone of inhibition.

One problem with both the FDA assay and the zone of inhibition assay is that it is not possible to distinguish whether antimicrobial agents cause cell death or cell stasis. However, it was found that this information could be obtained fairly easily by plating out the cultures from the FDA plate onto fresh agar.

Whilst this method is not as rigorous as the traditional broth dilution method

(Hattalin et al., 1973) it is simpler and uses far less resources. Nevertheless, it is only possible to detect a very high rate of cell death (>90%) using this method, which could explain the lack of consistency observed in the bactericidal activity of replicate samples.

Overall, the zone of inhibition assay and the FDA assay have provided valuable initial information about the antimicrobial activity in molluscan egg masses. These assays only require minimal amounts of sample making them suitable for screening a range of different species. The zone of inhibition assay provides a good preliminary assessment of the activity without the need for any major sample preparation. However, this method is not quantitative and can not be used to compare samples from different species. The FDA assay is a good method for rapidly screening extracts from different species. It can be used to determine minimum inhibitory concentrations and is suitable for bioassay directed purification of the active components (Chand et al.,

1995). The bactericidal assay provides additional information about the nature of the antimicrobial activity.

Chapter 4: Antimicrobial activity in molluscan egg masses 210

4.5.7 Epibiosis and symbiosis

Preliminary information on the presence of microorganisms associated with the surface of molluscan egg masses has been gained during this study.

Microbial growth was observed around all egg masses that were placed on marine agar. However, further experiments are required to confirm whether these microorganisms are epibiotic. Nevertheless, it is possible there is a commensal relationship between the egg masses of some molluscs and epibiotic microbes. Microorganisms derived from the surface of ten molluscan egg masses appear to competitively inhibit the growth of human pathogens in the zone of inhibition assay (Table 4.4). Many organisms are protected from pathogenic microorganisms as long as they retain their normal associated microflora (Austin, 1988; Gil-Turnes et al., 1989; Gil-Turnes and Fenical,

1992). Marine bacteria have also been found to produce inhibitory components against fouling organisms (Fenical, 1993; Holmström and

Kjelleberg, 1994). Gil-Turnes et al., (1989) have outlined an appropriate experiment to determine whether bacteria isolated from the surface of embryos provide chemical protection against pathogenic fungi.

The potential interactions between symbiotic microorganisms on the eggs of marine molluscs would provide an interesting area for further research. The use of bacteria as a source of biologically active compounds has clear advantages. The isolation and cultivation of some marine bacteria can be easily undertaken (Fenical, 1993; Holmström and Kjelleberg, 1994) and the collection of the active components produced by microbial cultures is usually

Chapter 4: Antimicrobial activity in molluscan egg masses 211 easy and inexpensive. This is an important factor when considering the difficulty in collecting the egg masses of some marine molluscs and the problems associated with cultivating marine macroorganisms. Furthermore, molecular techniques could be applied to marine bacteria to determine the genetic basis for the production of biologically active compounds (Holmström and Kjelleberg, 1994). Target genes could then be amplified and cloned into a host bacterium for large-scale commercial production.

It is possible that the antimicrobial activity found in the egg masses of some molluscs is derived from a bacterium associated with the substratum. Extracts from estuarine mud and intertidal pebbles were found to have antimicrobial activity in the FDA assay (Table 4.6). Samples of mud placed on agar produced similar microbial growth to the egg masses of five estuarine species

(Appendix 4.3), three of which incorporated mud into their egg ribbons (refer to Chapter 3). Interestingly, a number of biologically active marine microorganisms have been isolated from marine mud (Austin, 1988; Fenical,

1993). Bactericidal activity has also been reported in seawater and a number of seawater bacteria have been found to produce antibiotics (Fenical, 1993).

However, extracts taken from the seawater around Wollongong were not found to have antimicrobial activity (Table 4.6).

There are no culturing techniques which permit the recovery of all microbial cells associated with a sample from the marine environment (Austin, 1988). In fact it has been estimated that less than 5% of the bacteria observed in marine samples can be cultured under standard conditions (Fenical, 1993).

Chapter 4: Antimicrobial activity in molluscan egg masses 212

Therefore, it is important to note that foreign microorganisms from the marine samples were successfully competing with human pathogens, on nutrient agar at 370C, in this study. At least one microbe was also observed growing on marine agar at 250C. Colonies of marine microorganisms observed growing around samples on standard media could provide a good starting point for investigations into the presence of culturable epibiotic bacteria.

However, a range of other epibiotic microorganisms, which do not grow successfully under standard conditions, would not have been detected during this experiment. These could be investigated more thoroughly by microscopic methods.

4.6 Conclusion

In this study the gelatinous egg masses and leathery egg capsules from a wide variety of molluscs and several polychaetes were tested for antimicrobial activity.

Two complementary assays were used to screen the egg masses for antimicrobial activity; the zone of inhibition assay and the Fluorescein diacetate assay.

Antimicrobial activity was detected in the egg masses from 44 species, including two polychaetes and molluscs from a wide range of families. The results of this study confirm that the egg masses of marine invertebrates provide an interesting area for the discovery of potentially useful pharmaceuticals. Molluscan egg masses were found to inhibit the growth of a range of human pathogens and antimicrobial activity was also observed against several ecologically relevant marine pathogens. This provides support for the value of using a biorational approach to the discovery of novel sources of potential antibiotics.

Chapter 4: Antimicrobial activity in molluscan egg masses 213

The antimicrobial properties of the egg masses from several molluscs were lost during larval development. This suggests that there may be some form of chemical ripening in the egg masses. Antimicrobial activity is also restricted to the inner matrix of the egg masses from several species, rather than on the surface. Other species appear to contain epibiotic bacteria on the surface, which could potentially play a role in preventing infection from other bacteria. Foreign microbial growth was observed around all of the egg masses that were placed on marine agar, indicating that in general the egg masses are not protected from microbial fouling. This suggests that pathogenic infection of molluscan egg masses is a stronger selective pressure leading to the evolution of chemical defence, than microfouling on the surface of the egg masses.

NOTE

This online version of the thesis may have different page formatting and pagination from the paper copy held in the University of Wollongong Library.

UNIVERSITY OF WOLLONGONG

Chapter 5

Isolation and characterisation of the antimicrobial compounds from the egg mass of Dicathais orbita.

“Who has not heard how Tyrian shells

Enclosed the blue, that dye of dyes

Whereof one drop worked miracles”

Robert Browning, 1855.

5.1 Introduction

Tyrian Purple is most probably the first secondary metabolite to be isolated from a marine mollusc. As early as 1600 BC the people of Crete found a way to extract this purple dye from muricids (Baker 1974). Also known as Royal Purple and Purple of the Ancients, in Roman times Tyrian Purple was worth as much as 10-20 times its weight in gold (Baker 1974; Michel and McGovern 1990). The

Bible prescribes the use of purple and blue dyes, originally of molluscan origin, for colouring tabernacle curtains and priests vestiments (Exodus 26:1, 28;

Numbers 15:38; refer also to Hoffman, 1990; Elsner and Spanier, 1985).

The sociopolitical, religious and economic value of Tyrian Purple can be understood in terms of its rarity. There were few terrestrial sources of purple in

Roman times and most natural colours are not fast (Hoffmann, 1990). Pliny the

Elder stated that “the price of the dye depends on the yield of the coast”

(Bailey, 1929). Tyrian Purple could only be obtained from the hypobranchial Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 215

glands of a few species of marine molluscs and as many as 10,000 adults were required to produce just 1g of the dye (Friedländler, 1909; McGovern and

Michel, 1990; Michel et al., 1992). It is not clear whether the muricids were overharvested for the extraction of Tyrian Purple. However, the prices of purple dyed material rose steeply during the period it was most popular and by 300 AD the use of purple became restricted by laws and reserved for the Roman

Emperors and high ranking ecclesiastics (Baker, 1974; Hoffmann, 1990). This may have been the first law that was ever passed to protect a marine resource.

The commercial use of Tyrian Purple as a dye in the Mediterranean ceased in

1453 with the fall of Constantinople (Baker, 1974). A few centuries later Tyrian

Purple was rediscovered and by 1909, its chemical composition was elucidated by Friedländer as 6,6’-dibromoindigotin (5; Scheme 5.1. By this time there were already cheaper forms of synthetic purple available (Hoffmann, 1990).

Nevertheless, the historical importance of this dye has triggered much interest in the chemistry of its biosynthesis. Molluscs of the families Muricidae and

Thaisidae are the principal sources of the dye but no purple compound as such is present within the dye-producing glands of these molluscs. The original white fluid from the hypobranchial glands was found to turn yellow first, followed by various shades of green and blue to its final purple colouration, following exposure to sunlight (Cole, 1685). The colour change is associated with the production of an unpleasant sulfurous smell (Prota, 1980).

The ultimate precursor to the dye was isolated from an ethanol extract of the hypobranchial gland of the Australian murex, Dicathais orbita, by Baker and Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 216

Sutherland (1968). These researchers also isolated an enzyme from the glands, which initiated the conversion of the salt precursor (tyrindoxyl sulfate 1; Scheme

5.1), to the purple product. Subsequently, the intermediate precursors were isolated from diethyl ether extracts of the glands and identified as tyrindoxyl (2), which is oxidised to tyrindoleninone (3) (Baker and Duke, 1973a,b). These two compounds combine to form tyriverdin (4), the immediate precursor of Tyrian

Purple (5) (Chrisotophersen et al., 1978). Tyrindolinone (6), a methanethiol adduct of tyrindoleninone, together with 6-bromoisatin (7) have also been isolated in small amounts (Baker and Duke, 1973a; Baker, 1974). The 6- bromoisatin is a by product formed by the decomposition of tyriverdin and the amount produced increases relative to 6,6’-dibromoindigotin in the presence of oxygen (Baker, 1974). It has also been suggested that 6-bromoisatin may arise from the enzymatic oxidation of tyrindoxyl sulfate (1), the oxidation of tyrindoxyl

(2) and the hydrolysis of tyrindolinone (6) (Clark and Cooksey, 1997).

The role of the Tyrian Purple precursors in the hypobranchial glands of Muricid molluscs has remained a mystery (Prota, 1980; Max, 1989; Hoffmann, 1990).

Ziderman (1990) suggests that the “purple dyes are of no advantage to the sea snails”. This is because the dyes, as such, are not present in the living molluscs. It has been suggested that they merely represent excretory products from the breakdown of tryptophan (Fox, 1974; Ziderman, 1990). However, a function shaped by the process of natural selection seems more likely for these unusual metabolites (Prota, 1980). Several marine indoles have been shown to exhibit significant pharmacological activity (Christophersen, 1983) and in Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 217

particular, isatin, is a natural fungicide which protects the embryos of the shrimp, Lagenidium callinectes (Gil-Turnes et al., 1989).

There have been no previous investigations into the possible presence of Tyrian

Purple and its precursors in the egg masses of Muricid molluscs. Nevertheless, the egg masses of many species are yellow when freshly laid (Figure 5.1a) and turn a distinctive purple colour just prior to hatching (Figure 5.1b). Disruption of the egg capsules results in the liberation of an offensive sulfurous smell (pers. obs). Cole (1685) suggested that the products of the hypobranchial glands could be “ the spermatic and prolifick matter, by which they propagate their kind” (sic) but he was not able to conduct any experiments to test this idea.

Aristotle also indicated the potential importance of the egg masses by suggesting that the best time to collect the murex for their purple dye was before they have produced their ‘honeycomb-like exudate’ (Peck, 1970). Pliny the Elder reinforced this, stating that “this fact, although of utmost importance, is not recognised in the dye factories” (Bailey, 1929; Cap. XXXVIII. Sect. 62.

133.). Neither of these workers correctly identified this exudate as the egg masses. However, Bailey (1929) makes a note in his translation of Plinys’

Historia Naturalis that most molluscs envelope their eggs with a clear substance that is not unlike a honeycomb in appearance and indeed, the egg capsules of

Dicathais orbita bear a striking resemblance to these structures (Figure 5.1).

Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 218

Figure 5.1: The egg mass of Dicathais orbita a) fresh egg capsules under a dissecting microscope (10x magnification); b) an adult D. orbita amongst hatching egg capsules (0.75x).

Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 219

- + SO 3 X

SCH3

N Br Sulfatase H [Purpurase] 1. Tyrindoxyl sulfate

OH O

SCH3 SCH3 N Br N H Br

2. Tyrindoxyl 3. Tyrindoleninone

O O H SCH3 Br N SCH3

SCH N 3 N Br H Br H CH3S O 6. Tyrindolinone O 4. Tyriverdin 2 sunlight O

O H Br OH N N Br

N 7. 6-Bromoisatin Br H O 5. Tyrian Purple

Scheme 5.1: The formation of Tyrian Purple from the ultimate precursor tyrindoxyl sulfate produced in the hypobranchial glands of the muricid Dicathais orbita. Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 220

5.2 Objectives

In this component of the study, bioassay guided fractionation was used to isolate antimicrobial compounds from the egg mass of the abundant southern

Australian Muricid, Dicathais orbita. The egg mass of this species were also examined for the presence of Tyrian Purple and its precursors. The isolated compounds were identified and tested for bacteriostatic and cytolytic activity, against a range of human and marine pathogens, as well human lymphoma cells. This provided a preliminary examination of the potential pharmaceutical applications of the active metabolites and an assessment of their potential ecological role.

5.3 Methods

5.3.1 Collection and extraction of egg masses. The egg masses of Dicathais (Thais) orbita are common in the lower intertidal area from late winter through spring (personal observation). The eggs were collected from two sites, Towradgi and Flagstaff Hill (on the Illawarra Coast,

NSW, Australia), on several occasions throughout the breeding season (August to October) in 1995, 1996 and 1997. The eggs were collected from vertical rock faces by wading at Towradgi and by snorkelling at Flagstaff Hill. A total of 307 g of fresh eggs and 65 g of hatching eggs were collected. A voucher specimen

(M-em-001) is lodged in the Department of Biological Sciences, University of

Wollongong, NSW, Australia. Adults (108 g, 2 specimens) were also collected from Towradgi.

Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 221

The egg capsules were cut in half and extracted fresh by soaking in solvent for several hours, followed by decanting and replacing the solvent. This was repeated twice with the final soak being overnight. The extracts were then combined and evaporated to dryness using a rotary evaporator and water pump vacuum. The main solvent used was chloroform/methanol (1:1, v:v) but additional extracts were made using diethyl ether or ethanol. All solvents were

AR grade. A small volume of distilled water was added to the chloroform/methanol extracts to facilitate a chloroform, water/methanol separation before the extract was dried.

The adult molluscs were killed in the freezer and then the shell was removed by crushing in a vice. The whole bodies were extracted in chloroform/methanol according to the procedure outlined above.

5.3.2 Analysis of the crude extract The extracts from the egg masses of Dicathais orbita were analysed by thin layer chromatography on aluminium backed silica gel plates (Merck, 60 F254). A range of solvent systems were trialed for developing the chromatographs including dichloromethane (DCM, 100%), light petroleum/DCM (1:1),

DCM/methanol (9:1), ethyl acetate/petroleum ether (1:4), light petroleum/acetone (9:1) and benzene (100%). The best separations for the highly non polar compounds were obtained with light petroleum /DCM (1:1).

DCM (100%) was suitable for separating compounds with a slightly higher polarity. The chromatographs were observed in visible light, under UV light and developed in iodine vapour. Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 222

Water/methanol extracts from the fresh eggs of D. orbita were tested for the presence of tyrindoxyl sulfate by paper chromatography in butanol:acetic acid:water (78:5:17). The chromatographs were developed by spraying with a solution of vanillin (0.1% in ethanol) with 1M hydrochloric acid and then heating to 1000C.

Additional analysis was facilitated by mass spectrometry (MS) using a QP-5000

(Shimadzu) spectrometer with a direct insertion technique or by gas chromatography/mass spectrometry (GC/MS) using a GC-17A (Shimadzu) gas chromatograph in splitless mode. The injector temperature was set at 260oC.

The oven temperature was held at 40oC for 2 min then ramped to 290oC at a rate of 4oC per minute. The final oven temperature was then held at 290oC for

10 min. The carrier gas was helium and the flow rate was 1.4 ml/min. The electron beam energy in the mass spectrometer was 70eV and the source temperature was 200oC.

5.3.3 Isolation and Identification of Antimicrobial Components The isolation of antimicrobial compounds from extracts of the egg mass of

Dicathais orbita was bioassay directed, against Escherichia coli and

Staphylococcus aureus, using the fluorescein diacetate hydrolysis method (refer to Chapter 4). Chloroform extracts from the egg mass were fractionated by flash silica chromatography. The extract was passed through a silica column (25 g,

60 mesh) using redistilled DCM (200 ml) followed by 10% MeOH in DCM (v/v,

100 ml, redistilled) and 15 ml fractions were collected.

Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 223

The major components in the active fractions were isolated by radial chromatography on a Chromatotron (Professional Technology) using 9:1 light petroleum (bp 40-60 oC): dichloromethane (DCM) followed by 100% DCM, and then 5% methanol (MeOH) in DCM. All solvents were distilled prior to use and the solvent mixtures are expresses as volume to volume. The separation was repeated on fractions that still contained a mixture of two or more compounds.

The mass spectra of the isolated compounds were obtained by GC/MS, solid probe MS, as well as high resolution mass spectrometry (HR MS) using a

Fisons/VG Autospec-TOF-oa mass spectrometer. 1H nuclear magnetic resonance (1H NMR) spectra were determined at 300MHz with a Varian Unity-

300 spectrometer. The spectra were obtained on solutions in CDCl3 or DMSO- d6 and referenced to tetramethylsilane.

5.3.4 Antimicrobial testing Tests for antimicrobial activity were performed using the FDA assay (refer to

Chapter 4). The extracts and isolated compounds were tested for activity against the human pathogens Candida albicans (ACM4581, AMMRL 36.42,

AMMRL 36.70), Escherichia coli (ACM845), Staphylococcus aureus (ACM844) and Pseudomonas aeruginosa (ACM846), as well as two marine pathogens

Enterococcus sericolicida, Vibrio anguillarum and a mixed culture of skin flora

(refer to Chapter 4).

Weighed organic extracts or isolated compounds used in the assay were dissolved in acetone just prior to use. Water/methanol extracts were dissolved Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 224

in 20% acetone in MilliQ water. The samples were serially diluted from 1mg/ml to 0.0001mg/ml and at least three replicates were run at each concentration to determine the minimum inhibitory concentration (MIC). Crude extracts were also tested at 10mg/ml. Samples containing Tyrian Purple were placed in an ultrasonic bath prior to use, due to the insolubility of the compound in acetone.

After three hours incubation in the FDA assay 20µl of culture was plated out onto fresh agar to test for bactericidal activity (refer to Chapter 4). Cultures of cells incubated with the antimicrobial compounds were also observed under the light microscope. Slides were prepared by spinning 100µl of the culture onto the slides in a Cytospin 2 (Shandon, 150 r.p.m. for 3 mins). The slides were gram stained then viewed at 4x magnification and under oil on a light microscope

(100x).

5.3.5 Cytotoxicity testing Samples of tyriverdin were tested for cytotoxicity against human hystiocytic

0 lymphoma (cell line U937). The cells were grown at 37 C (5% CO2 atmosphere) in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS). The cells were harvested by centrifugation at 200 g for 5 min then resuspended in

RPMI/10% FBS (1x106 cells/ml) and aliquoted into a 96 well microplate (90

µl/well). Tyriverdin dissolved in acetone was diluted in RPMI/10% FBS and added to the cells (10 µl) at a range of concentrations (from 1 mg/ml to 0.001 mg/ml). Control cultures were incubated with acetone only. The cells were placed in an incubator at 370C and checked for evidence of cell death after two and six hours on a flow cytometer (FACSort, Becton Dickinson). Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 225

After incubation the cells were removed from the microtitre plate, resuspended in 1ml phosphate buffered saline (PBS, pH 7.4) and then centrifuged at 300 rpm for 5 min. This wash was repeated, then the cells were resuspended in PBS

(300µl). Propidium iodide (PI) was added (5 µg/ml final conc.) and the cells were analysed by FACSort using the Cell Quest software. Cells were analysed for viability based on the uptake of PI, with dead cells showing a greater uptake, which is indicated by increased FL2 fluorescence. Data from the side scatter, forward scatter and FL2 channels were collected. Statistics were collected by an arbitrary gating of cells, with anything below 102 on the FL2 scale considered viable.

5.4 Results

5.4.1 Analysis of the crude egg extracts. Thin layer chromatography revealed that the chloroform extracts from fresh, eggs contained a number of coloured compounds (Table 5.1). An orange spot

(Rf 0.5) was the major product present in the chloroform and ether extracts of the fresh eggs. A green spot (Rf 0.35), which turned purple in sunlight and a yellow spot (Rf 0.05) that did not change colour, were present in lipophilic extracts of both the fresh and hatching eggs (Table 5.1). In the ethanol extract, a yellow spot (Rf 0.4) that turned green and then purple in sunlight, was the dominant component. An insoluble purple spot, which remained on the base line, was the major component of the hatching eggs.

Paper chromatography of the water/methanol extract revealed a purple spot on the base line. No spot was observed around Rf 0.56, which was the Rf obtained Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbit 226

for tryindoxyl sulfate in the same solvent system (refer to Baker and Sutherland,

1968).

The major orange and green components of the fresh eggs were isolated successfully from a chloroform extract by silica chromatography. The yellow component broke down on silica to produce the orange and green components.

This is consistent with the results of Baker and Duke (1973) on the hypobranchial gland extractives, who identified the yellow component as tyrindolinone (6).

Table 1: Thin layer chromatographic analysis of the coloured compounds from various organic solvent extracts of Dicathais orbita eggs (with the solvent system light petroleum:dichloromethane (1:1); silica gel). The symbols used are: + for present, (+) present but minor component, and – for not detected.

Rf Colour Fresh Hatching

Chlorofor Ethano Diethyl Chloroform m l Ether 0.50 Orange + + + -

0.40 Yellow + + + -

0.35 Green + - + +

0.05 Yellow + - + +

0.00 Purple (+) - (+) + Chapter 5: Antimicrobial compounds from the egg mass of Dicathais orbita 227

The orange component (Rf 0.5) was identified as tyrindoleninone (3; Baker and

Duke, 1973) by GC/MS (Retention time 38.0 mins; M+• m/z 255, 257 79Br, 81Br; major fragments 240, 242 and 133 and 1H NMR spectroscopy (300 MHz,

CDCl3; δ2.65, s, 3H, CH3S ; δ7.3 – 7.4, m, 3H, ArH). HR MS (electron impact

+• 79 (EI)) produced an accurate mass M 254.9356 (C9H6 BrNOS requires

254.9353; the sample of tyrindoleninone also formed another high molecular weight product slowly at room temperature, although there was insufficient material to characterise it). Tyrindoleninone was found to be present in the fresh eggs but not in the hatching eggs using both the solid probe MS and GC/MS.

GC/MS revealed tyrindoleninone to be the major volatile organic constituent in the fresh egg masses (Figure 5.2).

The green component (Rf 0.35) in the egg mass was isolated by silica chromatography. The solid probe EI MS gave the closest match to Tyrian

Purple (5) in the mass spectrum library, with brominated isotopic ion clusters at m/z 418, 420, 422, as well as 257, 259 and 240, 242. HR MS (electron impact) gave an accurate mass for the high molecular weight fragment ion at m/z

79 417.8954 (C16H8 Br2N2O2 requires 417.8952). Previous studies have shown that tyriverdin (4) does not produce a molecular ion under electron impact conditions in the mass spectrum (Baker, 1967), although Christophersen et al.

(1978) has since shown that field desorption/ field ionization MS does produce a molecular ion. I investigated chemical ionization and electrospray (positive ion)

MS of this compound but still could not detect an MH+ ion. However, the 1H

NMR (DMSO-d6) of this green component demonstrated the presence of a singlet at δ1.9, corresponding to the SCH3 protons (6H), with additional peaks at Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 228

δ 8.2, s, 2H, NH; δ6.96, dd, J 8.4 Hz, 1.5Hz, 2H, ArH; δ7.27, d, J 1.5Hz, 2H,

ArH; δ7.46, d, J 8.4 Hz, 2H, ArH). This corresponds well to the values reported by Christophersen et al. (1978) for tyriverdin in the same solvent. The solid

+• probe MS indicated that tyriverdin (M -2CH3SH) was found in the hatching eggs, as well as the fresh eggs.

The polar yellow component (Rf 0.05), which did not change colour with time, was also isolated from the eggs by silica chromatography, using 5%MeOH in

DCM. The mass spectrum of this compound was matched to 5-bromoisatin in the GC/MS mass spectrum library (m/z M+• 225, 227 (79Br, 81Br); major fragment ions were observed at m/z 197, 199 and 170, 172). This is more likely to be 6-bromoisatin (7) as identified by previous research on the hypobranchial glands of muricids (Baker, 1974; Clark and Cooksey, 1997). HR MS (electron

+ 79 impact) gave an accurate mass for M at m/z 224.9423 (C8H4 Br NO2 requires

224.9425). 6-Bromoisatin was detected in trace amounts in both the fresh and hatching eggs using GC/MS.

Tyrian Purple was obtained by leaving pure tyriverdin in the presence of sunlight for several days. The product was then washed with ether and filtered to remove any remaining tyriverdin and 6-bromoisatin. The solid probe MS was used to confirm the absence of tyriverdin and 6-bromoisatin but the extreme insolubility of Tyrian Purple prevented spectra from being obtained on this compound.

Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 229

100%

tyrindoleninone

Intensity

6-bromoisatin

tyriverdin

Retention time (mins)

Figure 5.2: A GC/MS chromatogram of the volatile organic constituents in a chloroform extract from the egg masses of the Australian muricid, Dicathais orbita.

Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 230

5.4.2 Antimicrobial Activity Extracts from the egg masses of Dicathais orbita were found to exhibit significant antimicrobial activity, whereas the extracts from adult Dicathais orbita

(whole body) showed little activity (Table 5.2). Extracts of the fresh eggs were much more active than the hatching eggs and the extracts prepared with lipophilic solvents showed greater activity than a polar solvent extract (Table

5.2). Bioassay guided fractionation of the active compounds resulted in the isolation of three antimicrobial compounds, which were identified as

Tyrindoleninone, Tyriverdin and 6-Bromoisatin. The minimal inhibitory concentration of both the crude extracts and isolated compounds was similar against a range of different human and marine pathogens (Table 5.2; 5.3).

In the FDA assay, tyriverdin was the most active compound isolated from the egg masses (Table 5.2). Antimicrobial activity was observed at a concentration of 1-0.5 µg/ml against all test microorganisms for tyriverdin, which was a substantially greater activity than the crude extract. Tyrindoleninone was mildly bacteriostatic against both human and marine pathogens, whereas 6-

Bromoisatin did not effectively inhibit the growth of two marine bacteria (Table

5.2). Tyrian Purple exhibited minimal activity, which could be due to trace amounts of tyriverdin or 6-bromoistatin left in the sample. It should be noted however, that a sonicated suspension of Tyrian Purple had to be used in this assay due to the insolubility of the compound in acetone.

Plating out the cultures from the FDA assay revealed that tyriverdin was only bacteriostatic and lysed less than 90% of the microbial cells at a concentration Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 231

of 1 mg/ml (Table 5.3). This was confirmed under the light microscope where the cells were seen to cluster around crystals of tyriverdin but only those cells in contact with the crystals were lysed or distorted (Figure 5.3; 5.4; 5.5). The cells in cultures of the Gram negative marine pathogen Vibrio anguillarium appeared purple in comparison to the control cells after incubation with tyriverdin (Figure

5.3). The yeast Candida albicans appeared to be most susceptible to cell lysis by tyriverdin (Table 5.3). Under the light microscope cultures of Candida albicans incubated with tyriverdin (1 mg/ml) were greatly reduced in density compared to the control cultures and most of the cells appeared elongated and shrunken, whilst others appeared swollen (Figure 5.4). Notably, the crystals of tyriverdin remained green under the light microscope after at least 12 hrs incubation with the cells.

Tyrindoleninone was highly toxic to the cells at a concentration of 1 mg/ml, causing 100% cell death to most of the microorganisms (Table 5.3). Only a few patches of lysed or distorted cells were observed under the light microscope for cultures of Staphylococcus aureus, Escherichia coli (Figure 5.3), Vibrio angullarium (Figure 5.3) and Candida albicans (Figure 5.5) after incubation with tyrindoleninone. In the mixed culture of skin flora a few Gram positive cocci remained after incubation with tyrindoleninone but no Gram positive rods were observed (Figure 5.4). Cultures of the Gram positive marine pathogen

Enterococcus sericolicida and the Gram negative bacterium Pseudomonas aeruginosa were more resistant to tyrindoleninone than the other test organisms

(Table 5.3). However, incubation with tyrindoleninone reduced the density of cells in cultures of these two bacterium incubated, compared to control cultures. Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 232

Under the light microscope, pink patches of lysed cells were clearly visible, in addition to normal purple looking cells (e.g. Figure 5.4).

The antimicrobial activity of 6-bromoisatin was not much greater than the crude extract (Table 5.2; 5.3). Some evidence of cell death was apparent from plating out cultures of the Gram positive bacterium Staphylococcus aureus and the

Gram negative bacterium Vibrio angullarium after incubation with 6-bromoisatin

(Table 5.3). However, under the light microscope some lysed cells were observed in cultures of all the test microorganisms that had been incubated with

6-bromoisatin (Figure 5.3; 5.4; 5.5). The density of cells in the cultures of

Candida albicans was significantly reduced by 6-bromoisatin, although the remaining cells did not appear distorted or swollen (Figure 5.5). Notably, cells of the Gram negative bacteria Vibrio angullarium appear Gram positive (purple) when in contact with crystals of 6-bromoisatin (Figure 5.3).

5.4.3 Cytotoxicity of tyriverdin There was no evidence of any increase in cell death in populations of human hystiocytic lymphoma cells (cell line U937) after two or six hours incubation with tyriverdin at a range of different concentrations (Table 5.4).

Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 233

Table 5.2: Bacteriostatic activity of the extracts (ex.) and isolated compounds from the egg masses and adult tissue of Dicathais orbita. The test microorganisms are Staphylococcus aureus (S.a.), Escherichia coli (E.c.), Pseudomonas aeruginosa (P.a.), Candida albicans (C.a.), Enterococcus sericolicida (E.s.), Vibrio angillarium (V.a.) and a mixed culture of skin flora (Mixed).

Solvent extract Minimum Inhibitory Concentration (mg/ml) or compound S.a. E.c. P.a. C.a. E.s. V.a. Mixed

Adult tissue > 10 - - NT NT NT NT Chloroform ex. Fresh eggs 0.1- 1 0.1-1 1-10 0.1-1 1 1 1 Chloroform ex. Fresh eggs 0.1 0.1 0.1 NT NT NT NT Diethyl ether ex.

Fresh eggs 10 10 10 NT NT NT NT Ethanol ex.

Fresh eggs ------Methanol/Water ex.

Hatching eggs 10 10 10 NT NT NT NT Chloroform ex.

Tyriverdin 0.0005 0.0005 0.0005 0.001 0.001 0.001 0.001

Tyrindoleninone 0.5 0.5 1 0.1 0.1 0.1 0.1

6-Bromoisatin 0.1 1 1 >1 >1 >1 >1

Tyrian Purple >1 >1 >1 >1 1 1 1

NT not tested

- no activity at the maximum concentration tested (50mg/ml for

water/methanol extracts, 10mg/ml for the other extracts and 1mg/ml for

isolated compounds).

> partial activity at the maximum concentration tested. Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 234

Table 5.3: The concentration of the extracts (ex.) and compounds isolated from the egg masses of Dicathais orbita required to cause 100% cell death. The test microorganisms are Staphylococcus aureus (S.a.), Escherichia coli (E.c.), Pseudomonas aeruginosa (P.a.), Candida albicans (ACM4581; C.a.), Enterococcus sericolicida (E.s.), Vibrio angillarium (V.a.) and a mixed culture of skin flora (mixed).

Sample Minimum concentration to elicit 100% cell death (mg/ml)

S.a. E.c. P.a. C.a. E.s. V.a. mixed

Fresh eggs >10 >10 - >10 >10 >10 10 Chloroform ex.

Tyriverdin - - - >1 - - -

Tyrindoleninone 1 1 >1 1 >1 1 1

6-Bromoisatin >1 - - - - >1 -

Tyrian Purple ------

> indicates partial activity at the maximum concentration tested (10mg/ml for crude extract and 1mg/ml for isolated compounds).

- indicates no detectable activity at the maximum concentration tested. It should be noted, however, that this does not exclude the potential for some cell death because this assay will only detect a high level of cell death (ie. 90-100% cell death).

Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 235

Figure 5.3: The gram negative bacteria Escherichia coli (left panels) and the marine pathogenVibrio anguillarum (right panels) viewed at 100x magnification under a light microscope after incubation with compounds isolated from the egg mass of Dicathais orbita at 1mg/ml: a) acetone only (i.e. control); b) tyrindoleninone; c) tyriverdin; d) 6-bromoisatin. Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 236

Figure 5.4: Gram positive bacteria from a mixed culture of skin flora including Coryrebacterium sp., Lactobacillus sp. and Staphylococcus sp. (left panels) and the marine pathogen Enterococcus seriolocida (right panels) viewed at 100x magnification under a light microscope after incubation with compounds isolated from the egg mass of Dicathais orbita at 1mg/ml: a) acetone only (i.e. control); b) tyrindoleninone; c) tyriverdin; d) 6-bromoisatin. Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 237

Figure 5.5: Cultures of Candida albicans (AMMRL 36.70) viewed under the light microscope at 10x magnification (left panels) and 100x magnification (right panels) after incubation with compounds isolated from the egg mass of Dicathais orbita at 1mg/ml; a) acetone only (i.e. control); b) tyrindoleninone; c) tyriverdin; d) 6-bromoisatin. Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 238

Table 5.4: The proportion of human cells remaining alive after six hours incubation with tyriverdin at a range of different concentrations and compared to a control culture incubated with acetone only.

Sample Percent live cells

Acetone control 95.8

Tyriverdin 1mg/ml 96.9

Tyriverdin 0.1mg/ml 96.6

Tyriverdin 0.01mg/ml 96.9

Tyriverdin 0.001mg/ml 97.2

Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 239

5.5 Discussion

5.5.1 Antimicrobial compounds from the egg mass of Dicathais orbita The egg masses of Dicathais orbita were found to contain the well known dye

Tyrian Purple (5) and several of its precursor compounds. The demonstrated antimicrobial activity of the precursors, tyrindoleninone (3) and tyriverdin, (4), against both human and marine pathogens, suggests that these compounds may be produced by Dicathais orbita to keep the developing embryos free from infection. The sterilisation of brood cells has previously been demonstrated for terrestrial invertebrates, such as honey bees (Blum et al.,

1959) and a number of marine molluscs are known to protect their eggs with natural products (e.g. Paul and Van Alstyne, 1988; Pawlik, et al., 1988; Paul and Pennings, 1991). However, this is the first report of antimicrobial compounds in the brood cells of a marine mollusc. This is also the first study to provide evidence of a natural function for these Tyrian Purple related compounds.

The pattern of antimicrobial activity exhibited by the brominated compounds isolated from the egg masses of Dicathais orbita corresponds well to their presence during the different stages of egg development. Tyrindoleninone (3) is the major constituent present in the fresh eggs of D. orbita and was found to have the greatest toxic effect on the bacterial cells. During egg development tyrindoleninone (3) is converted to tyriverdin (4), which was found to have stronger antimicrobial activity but was not as toxic to the microbial cells. By the time the Dicathais embryos hatch, the egg capsules have turned purple (Figure 5.1). This indicates that most of the tyriverdin Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 240 present has been converted into Tyrian Purple (5), which does not appear to have significant antimicrobial activity. The loss of antimicrobial activity during development of D. orbita eggs may be the mechanism by which the embryos are protected from autotoxicity.

It appears that the precursors of Tyrian Purple are present in sufficient concentrations in the egg masses of Dicathais orbita to realistically reduce the chances of microbial infection. Both thin layer chromatography and GC/MS indicate that these compounds dominate the organic extracts and tyriverdin, at least, is active at extremely low concentrations (0.1µg/ml). Nevertheless, quantitative data is required to fully understand the role of these secondary metabolites in the egg masses. In particular, future studies should determine how the concentrations of the different precursors vary over embryonic development. However, in this study, quantification of the different metabolites was hampered by the fact that tyrindolinone and tyriverdin decompose in air and light respectively, as well as the insolubility of Tyrian Purple.

A number of other Muricid molluscs are known to produce Tyrian Purple

(Baker, 1974) but the egg masses of only one species, Nucella lapillus, have been tested previously for antimicrobial properties. In fact the intracapsular fluid of N. lapillus was reported not to possess antimicrobial activity against 13 strains of bacteria (Pechenik et al., 1984). However, the hypobranchial secretions of Nucella lapillus are known to contain precursors of Tyrian purple

(Clark and Cooksey, 1997). Furthermore, the egg capsules of Nucella lapillus turn purple in sunlight (personal observation), which suggests that like D. Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 241 orbita, they too contain Tyrian Purple and its precursors. Consequently, the egg masses of N. lapillus would be expected to exhibit some antimicrobial activity, unless they were tested at a late stage of embryo development, as demonstrated for the hatching eggs of Dicathais orbita. The lack of observed antimicrobial activity in the egg masses of Nucella lapillus could also be explained by inappropriate methodology (refer to Chapter 4).

It is not presently known how the precursors of Tyrian Purple get to the egg mass from the hypobranchial gland of the adult, where the ultimate precursor tyrindoxyl sulfate (1) and the sulfatase enzyme, are produced (refer to Baker and Sutherland, 1968). However, the oviduct of neogastropods does extend into the mantle cavity where the hypobranchial gland is located (Kay et al.,

1998). The capsule and albumen glands, where the egg capsules are elaborated and the fertilised eggs are encased, are also located within the mantle cavity (Kay et al., 1998). Further anatomical studies are required to establish a link between the hypobranchial gland and the oviduct in Muricids.

In this study, extracts from the whole bodies of Dicathais orbita were not found to have antimicrobial activity (Table 5.2). However, the hypobranchial glands of muricids have been shown to exhibit significant neuromuscular blocking activity (Welsh, 1964; Whittaker, 1960; Roseghini et al., 1996). A mixture of choline esters appear to be responsible for this pharmacological activity (Whittaker, 1959; Duke et al., 1981; Roseghini et al., 1996). It is therefore of interest that the ultimate precursor of Tyrian Purple is held as a choline ester salt in the hypobranchial glands of Muricids (Baker and Duke, Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 242

1976; Duke et al., 1978). The physiological function of these esters is uncertain (Whittaker, 1960), although it is possible that they are constituents of the venom used by these predatory gastropods to paralyse their prey

(Roseghini et al., 1996). Nevertheless, it is apparent that the hypobranchial gland is an organ adapted for the production and storage of biologically active substances.

5.5.2 Tyriverdin- a novel drug lead Tyriverdin provides a useful lead for the design and synthesis of new antimicrobial agents. The minimum inhibitory concentration found for tyriverdin in this study was lower than that reported for penicillin G using the same antimicrobial assay (Chand et al., 1995). Tyriverdin has broad spectrum activity against a range of human pathogens (Table 2.2) but is not toxic to human cells (Table 2.3). In this sense tyriverdin is similar to the aminoglycoside, cephalosporin, chloramphenicol and penicillin antibiotics

(refer to Cannon, 1995). It is also of interest that tyriverdin appears to be bacteriostatic at low concentrations but bactericidal at higher concentrations

(Table 5.2; Figure 5.3; 5.4; 5.5). These features are characteristics of the lincosamides and macrolides, which inhibit protein synthesis in bacterial cells

(Cannon, 1995).

The lack of antimicrobial activity in Tyrian Purple, as compared to tyriverdin, indicates that the methylthio groups could be important for the observed bacteriostatic activity. Organo-sulfur compounds form a major therapeutic resource and in fact, many classes of drugs contain compounds with sulfur in Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 243 their structures (Mitchard, 1988). In particular, sulfur-containing dyes have given rise to many clinically useful substances (sulphonamides) and the beta lactam antibiotics are naturally occurring sulfur containing molecules

(Mitchard, 1988; Cannon, 1995). Many thiols and aromatic disulphides are believed to be toxic because they produce thiyl radicals and “active oxygen” species (Munday, 1985; 1989; Munday and Manns, 1985).

In order to gain some assessment of the three dimensional structure of tyriverdin, preliminary computer-aided molecular modelling studies were undertaken, in vacuo (Griffith, pers. comm.). A systematic conformational search was performed around the central single bond of both the RR’ and RS’ isomers of tyriverdin using the SPARTAN modelling program. Rotation was done in 600 increments and the conformations were optimised using molecular mechanics. The conformations were then reoptimised at a semiempirical level. These studies have revealed five possible conformations for both the RR’ and RS’ isomers (Figure 5.6). The lowest energy conformations of tyriverdin were found to be folded structures, with a distance of only 2.39-2.44 Å between the two sulfur atoms (Figure 5.6). These distances are significantly smaller than the accepted van der Waals contact distance between two sulfide groups (3.70 Å; refer to Rosenfield and

Parthasarathy, 1974). This suggests that the three dimensional structure of tyriverdin could be stabilised by nonbonded intramolecular interactions between the sulfur groups. By comparison, significantly short contact distances between the sulfur and nitrogen atoms were found in other conformations of tyriverdin (Figure 5.6). Intramolecular dipole-dipole Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 244 interactions have been described in a number of other antibiotics, between sulfur and several electronegative atoms, including sulfur, oxygen and nitrogen (Nagao et al., 1998). On the basis of X-ray crystallographic studies on the compounds, these authors reported bond lengths between 2.03 and

3.25 Å for the non-bonded atoms.

There appears to be no previous studies reporting intramolecular nonbonded

1,4-type S…S interactions, such as that predicted for tyriverdin (Figure 5.6).

However, a 1,5-type S…S interaction has been described in four synthetic

… compounds (Nagao et al., 1998) and an unusually short S S contact distance has been found in the crystal structure of the naturally occurring amino acid meso-lanthionine dihydrochloride (Rosenfield and Parthasarathy; 1974). It is also of interest that nonbonded 1,4-type S…O interactions in the thiazole nucleosides are thought to play an important role in the mechanisms of several biological effects, such as antitumor activity and enzyme inhibition

(Goldstein et al., 1988; Goldstein et al., 1994; Franchetti et al., 1995).

Hydrogen bonds may also play a role in the stabilisation of the conformations of tyriverdin. In (RR’)- tyriverdin, potential hydrogen bonds were found between the oxygen atoms and the hydrogen of the amine moieties in most of the folded conformations. Higher energies were associated with the conformations that were stabilised by fewer hydrogen bonds (Figure 5.6). The folded RS’ conformations have high energies relative to the folded RR’

… conformations and these were only found to have SCH3 O hydrogen bonding. Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 245

Hydrogen bonding was also observed in the relatively planar conformations of tyriverdin, in which the SCH3 groups are not eclipsed (Figure 5.6). The highest energy is associated with the non-eclipsed RR’ isomer, which was only found to have a hydrogen bond between the oxygen atoms and a hydrogen atom on the methylthio moiety (Figure 5.6). By comparison, the non-eclipsed conformations Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 246

Figure 5.6: The minimum energy conformations of RR’ tyriverdin (right)

and RS’ tyriverdin (left). Sulfur atoms are shown in green, nitrogen in blue,

oxygen in red, bromine in brown, carbon in black and hydrogen in grey.

Significant intramolecular interactions are represented using dashed lines.

Nonbonded atoms distances of less than 3.2 Å were taken to indicate

dipole-dipole interactions between two sulfur atoms (shown in black) or

between sulfur and nitrogen atoms (shown in blue). Nonbonded atoms

distances less than or equal to 2.5 Å were taken to indicate hydrogen

bonding (shown in red). The energy associated with each conformation

(kc/mol ≡ kcal/mol) is provided underneath the structure. Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 247

RR’ Tyriverdin RS’ Tyriverdin

45.21 kc/mol 52.12 kc/mol

45.23 kc/mol 53.00 kc/mol

46.33 kc/mol 53.00 kc/mol

47.76 kc/mol 53.99 kc/mol

58.54 kc/mol 53.99 kc/mol Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 248

of (RS)-tyriverdin had lower energies and these appeared to be stabilised by

NH…O intramolecular hydrogen bonds (Figure 5.6). X-ray crystallographic studies are required to determine which of the three possible stereoisomers of tyriverdin (a pair of enantiomers and one meso form) are naturally occurring and to ascertain the preferred conformation in the solid state.

Tyriverdin is an unstable compound and it is possible that the decomposition products are responsible for the observed antimicrobial activity. In the presence of sunlight tyriverdin undergoes radical cleavage to form Tyrian

Purple (5) and dimethyl disulfide (Scheme 5.2; Baker and Sutherland, 1968).

Dimethyl disulfide is known to result in toxicity in ruminants (Steven et al.,

1981; Carlson and Breeze, 1984) and dimethyl sulfide can be toxic to rats

(Butterworth et al.,1975). Nevertheless, the formation of Tyrian Purple and the concomitant liberation of dimethyl disulfide is unlikely to be responsible for the antimicrobial activity of tyriverdin detected in this study, because the assays were carried out in the dark.

In the absence of sunlight it is possible that tyriverdin could undergo some elimination at the hemithioaminal-type moiety to form dehydrotyrian purple

(anhydro-6,6’-dibromoindigotin, 8) and methane thiol (Scheme 5.2). Methane thiol is known to be lethal to rats in air at a concentration of 10,000ppm

(Spencer, 1956) but the antimicrobial properties of this compound have not been reported. The biological activities of dehydrotyrian purple have also not been reported. Nevertheless, it is significant that the crystals observed in bacterial cultures incubated with tyriverdin appeared green under the light Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 249

O H SCH3 Br N

N Br H CH3S O Sunlight

O - 2 CH3SH H Br N

• N Br H SCH3 O • SCH3 O Br N - CH3SSCH3

N Br

O O H R 8. Dehydrotyrian Purple N

N R H O 5. Tyrian Purple

Scheme 5.2: The decomposition of tyriverdin by radical cleavage in the presence of sunlight or elimination in the absence of sunlight. Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 250 microscope. The decomposition of tyriverdin would be expected to yield purple crystals if Tyrian Purple was formed, or red/ brown crystals if dehydrotyrian purple was formed (Christophersen et al., 1978).

Overall, the antimicrobial and chemical properties of tyriverdin make it a useful lead for drug development. The uncomplicated structure of tyriverdin means that both it and its analogues can be synthesised for future research.

Methods for the synthesis of Tyrian Purple and tyriverdin have been described

(Friedländer et al., 1906; Christophersen et al., 1978; Pinkney and Chalmers,

1979; Gerlach, 1991). Indeed, future research should use synthetic tyriverdin to obviate further harvesting of this important reproductive stage of Dicathais orbita from the field.

Research on the structure/activity relationships of tyriverdin is currently under investigation, involving the synthesis of analogues and further molecular modelling studies (Torkamani et al., 1998). These studies could provide further insight into the involvement of intramolecular dipole-dipole interactions in the inhibition of bacterial enzymes.

5.6 Conclusion

Three antimicrobial compounds have been isolated from the egg mass of the common southern Australian Muricid, Dicathais orbita (Gmelin, 1791). These compounds are shown to be brominated indoles and precursors to the ancient dye Tyrian Purple. The fresh egg masses were found to contain a high proportion of tyrindoleninone, which reacts to form tyriverdin and Chapter 5. Antimicrobial compounds from the egg mass of Dicathais orbita 251 subsequently Tyrian Purple and 6-bromoisatin as the eggs develop and the larvae hatch. Antimicrobial testing revealed that tyrindoleninone is toxic to a range of human and marine pathogens at a concentration of 1mg/ml.

Tyriverdin is effectively bacteriostatic at 0.0005mg/ml but does not appear to be strongly bactericidal or toxic to human lymphoma cells. The chemical structure and antimicrobial properties of tyriverdin make it a useful lead for drug synthesis. The 6-bromoisatin was found to have mild antimicrobial properties, whereas Tyrian Purple exhibited no significant activity. The antimicrobial properties of these compounds and changes in their presence during egg development correlates with ripening in the egg masses of

Dicathais orbita. This is the first report of the chemical ripening of eggs in the marine environment.

NOTE

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UNIVERSITY OF WOLLONGONG

Chapter 6:Volatile organic compounds in molluscan egg masses 252

Chapter 6

Volatile organic compounds in molluscan egg masses.

6.1 Introduction

The difficulties associated with collecting organisms from the marine environment and the lack of ethnomedical history has meant that marine natural products research lags a long way behind the equivalent research on land

(Faulkner, 1992; Carté, 1996). It has been suggested that the marine environment is a major untapped source of biologically active compounds

(Flam, 1995; Carté, 1996; Cragg et al., 1997). Only a small proportion of marine species have been chemically studied in detail and the marine environment has a vast biological diversity, with a broader range of taxonomic levels compared to the terrestrial environment (de Vries and Hall, 1994). It is expected that chemical diversity should be, to some degree, proportional to biological diversity

(de Vries and Hall, 1994; Carté, 1996). Secondary metabolites are characterised by their heterogeneity and a restricted distribution, occurring in only some groups or species (Luckner, 1984; Avila, 1995). Consequently chemical diversity in the marine environment could exceed the diversity of biologically active compounds found on land.

Many of the bioactive compounds that have been isolated from marine organisms exhibit structural features that are not found in terrestrial natural products (Fenical, 1982; Carté, 1996). In general, the biosynthetic pathways used to produce secondary metabolites are similar in the marine and terrestrial Chapter 6: Volatile organic compounds in molluscan egg masses 253

environment. However, the composition of marine secondary metabolites can be significantly modified by virtue of the composition of the available elements in the sea (Fenical, 1982). The sea is a halide rich environment, which has allowed marine organisms to incorporate bromine, chlorine and iodine into covalent organic structures. Sulfur is also present in high concentrations in seawater, mainly as sulfate and in reducing sediments as sulfide. Nevertheless, this element is only infrequently encountered in marine secondary metabolites in the reduced organic form (Fenical, 1982).

A good correlation has been found between antimicrobial activity and halogen content in a range of marine organisms (Shaw et al., 1974). Marine organisms incorporate halogens into a wide range of chemical structures, including terpenes, alkaloids, acetogenins and amino acids. Halomethanes, which are potent biocides, have been isolated from red seaweed along with a large number of other small halogenated metabolites (McConnell and Fenical, 1977).

Halogenated indole residues, formed from the degradation and transformation of the amino acid tryptophan, are also frequently found in marine organisms

(Fenical, 1982; Christophersen, 1983; Alvares and Salas, 1991; Davis and

Bremner, in press).

Amongst the mollusca, Shaw et al. (1974) found a particularly strong positive relationship between bromine content and antimicrobial activity.

Bromoperoxidase, an enzyme which catalyses the oxidation of bromide and resulting in the bromination of a suitable substrate, was first isolated from a marine red alga (Theiler et al., 1978). Brominating enzymes have been isolated Chapter 6: Volatile organic compounds in molluscan egg masses 254

subsequently from a wide range of marine algae and prokaryotes (Ahern et al.,

1980; Baden and Corbett, 1980; Van Pe’e and Lingens, 1985a;b). However, bromoperoxidase activity has only been demonstrated in one mollusc, the

Muricid Murex trunculus (Jannun and Coe, 1987). The occurrence of brominated indoles has been well documented in the muricids (Baker, 1974;

Prota, 1980; Christophersen, 1983; Chapter 5).

6.1.1 Indole derivatives from muricids Two precursors of Tyrian Purple (6,6’-dibromoindigotin, 5), were found to be responsible for antimicrobial activity in the egg mass of the Australian Muricid

Dicathais orbita (Chapter 5). Antimicrobial activity has been demonstrated in the egg masses of five muricids, in addition to Dicathais orbita (Chapter 4). The production of Tyrian Purple from precursors in the hypobranchial has been demonstrated from a number of species of muricids (Baker, 1974; Prota, 1980;

Christophersen, 1983). Consequently, the same compounds could be responsible for the antimicrobial activity in the egg masses of the different species of Muricidae.

Investigations on the hypobranchial glands of several murcids have revealed that the number and nature of precursors involved in the production of Tyrian

Purple differs between species (Fouquet and Bielig 1971; Baker, 1974; Baker and Duke, 1974; Prota, 1980). Only one ultimate precursor tyrindoxyl sulfate (6- bromo-2-methylmercaptoindoxyl-3-sulfate, 1; Chapter 5, Scheme 5.1) has been isolated from the hypobranchial gland of Dicathais orbita (Baker and

Sutherland, 1968). However, Fouquet and Bielig (1971) have reported four Chapter 6: Volatile organic compounds in molluscan egg masses 255

chromogens from the glands of the Mediterranean muricid Trunculariopsis

(Murex) trunculus (9, 10 Scheme 6.1; 16, 17 Scheme 6.2). Chromogen 17 was thought to be the sole precursor to Tyrian Purple in the glands of Ceratostoma

(Murex) erinaceus and two other Mediterranean muricids (Prota, 1980).

However, Baker (1974) has suggested that these species contain two purple precursors, including the additional chromogen 18 from Purpura haemastoma.

The different chromogens found in the hypobranchial glands of muricid molluscs react by two different pathways to produce a suite of intermediate precursors to the final indigoid dyes (Prota, 1980). The chromogens 9 and 10 were found to react anaerobically in the dark with a sulfatase enzyme to yield indoxyl (13) and

6-bromoindoxyl (14) respectively (Scheme 6.1). These then react aerobically in the dark to afford the corresponding indigoid dyes (15 and 5). On the other hand, like tyrindoxyl sulphate, the chromogens 16 and 17 react aerobically with sulphatase in the dark to give the corresponding indoxyl (20 and 22) and green tyriverdin type products (23 and 24; Scheme 6.2; refer to Chapter 5, Scheme

5.1). These undergo photolytic cleavage in sunlight to give the same dyes (15 and 5). Consequently, different indole intermediates could be found in the egg masses of different species of muricids.

The production of indigotin (15) in addition to Tyrian Purple (6,6’ dibromoindigotin; 5) has been reported from the hypobranchial glands of only one muricid, Trunculariopsis (Murex) trunculus (Elsner and Spanier, 1985;

Michel et al., 1992). In addition to these two dyes, T. trunculus was found to produce a suite of other purple products (Baker, 1974), including 6- Chapter 6: Volatile organic compounds in molluscan egg masses 256

bromoindigotin (25), a cross reaction product of the indoxyls and 6- bromoindoxyls (Michel et al., 1992). Surprisingly, 6-bromoindigotin (25) has also been found in Tyrian Purple extracts from Nucella lapillus, despite the detectable absence of any indigotin (Clark and Cooksey, 1997).

The presence of indirubins (26-29), isomers of the indigoids, has also been demonstrated from the secretions of M. trunculus (Michel et al., 1992) and 6,6’- dibromindirubin (26) is present as a minor constituent in the secretion of N. lapillus (Clark and Cooksey, 1997). The bromoindirubins are thought to form from the condensation of tyrindoxyl with 6-bromoisatin (Clark and Cooksey,

1997). As 6-bromoisatin is likely to be an oxidation product of tyriverdin (Baker,

1974) a greater yield of indirubins could be expected under aerobic conditions.

Overall, the photochemical and oxidative reactions of the muricid dye precursors could create problems for distinguishing the actual components present in the egg masses from artefacts of the extraction and preparation procedures.

6.1.2 Other bioactive halogenated compounds from molluscs A number of bioactive halogenated compounds have been isolated from sea hares in the genus Aplysia. These are primarily terpenes and include a wide range of brominated and chlorinated, mono-, di- and sequiterpenes (Faulkner,

1984a; 1992a and references therein). Other bioactive halogenated compounds isolated from Aplysia spp. include the acetogenin Aplyparvunin (Miyamoto et al.,

1995), a range of C15 acetylenic ethers (Vanderah and Schmitz, 1976) and the Chapter 6: Volatile organic compounds in molluscan egg masses 257

- + SO3 X

9. R=H N R H 10. R=Br Sulfatase

OH O

R N N H R H 11. R=H 13. R=H O 12. R=Br 2 14. R=Br O H N R

N R H O 15. R=H; Indigotin Scheme 6.1 5. R=Br; 6,6’- Dibromoindigotin

- + SO3 X

R’ Sulfatase N 16. R=H, R’=SCH3 R H 17. R=Br, R’=SO2CH3 18. R=H, R’= SO2CH3

OH O

O2 R’ R’

R N N H R H

19. R=H, R’=SCH3 21. R=H, R’=SCH3 20. R=Br, R’=SO2CH3 22. R=Br, R’=SO2CH3 O R’ H N R

N R H R’ O Sunlight 23. R=H, R’=SCH3 15 24. R=Br, R’=SO CH 5 Scheme 6.2 2 3 Chapter 6: Volatile organic compounds in molluscan egg masses 258

O H N

N Br H O 25. 6-Bromoindigotin

O O

NH NH N N H Br H O O

26. 6,6’-Dibromoindirubin 27. Indirubin

O O

NH NH N N Br H H O O

28. 6-Bromoindirubin 29. 6’-Bromoindirubin Chapter 6: Volatile compounds in the egg masses of molluscs 259

aplysiatoxins, which are complex phenolic lactones (Kato and Scheuer, 1974).

All of these metabolites are primarily found in the digestive glands and are directly related to constituents of their algal diets (Faulkner, 1984a; 1992).

However, the egg masses of Aplysia spp. do not appear to contain the sequestered metabolites found in the adult sea hares (Faulkner, 1992;

Pennings and Paul, 1993).

Diet derived halogenated compounds have also been isolated from a number of other Opisthobranchs. The notaspidean mollusc Tylodina fungina is always found feeding on sponges in the genus Aplysina and contains the same brominated metabolites as its food source (Faulkner, 1992). Similarly, extracts of the dorid nudibranch Chromodoris funerea yielded a polybrominated metabolite, which was found in its host sponge Dysidea herbacea (Carté et al.,

1986). The nudibranch Phestilla melanobranchia contains a series of brominated indole alkaloids that are derived from its food source, hard corals in the genus Tubastrea (Okuda et al., 1982). Four unusual chlorinated diterpenes have been isolated from the nudibranch Chromodoris hamiltoni (Pika and

Faulkner, 1995) and a series of chlorinated acetylenes were isolated from one collection of Diaulula sandiegensis (Walker and Faulkner, 1981). The source of these metabolites is not known. However, it is likely that these are also dietary derived because there is no record of any chloroperoxidase enzymes being found in molluscs. One brominated and chlorinated terpene has been isolated from the bubble shell Haminoea cymbalum (Poiner et al., 1989). This metabolite was not found in the alga on which the bubble shell was found, although this does not exclude the possibility of a dietary source. Chapter 6: Volatile compounds in the egg masses of molluscs 260

6.1.3 Nonhalogenated bioactive compounds from molluscs Despite the reported correlation between halogen content and antimicrobial activity by Shaw et al., (1974) most of the biologically active compounds that have been isolated from marine molluscs do not appear to be halogenated. A large range of predominantly lipophylic bioactive compounds have been identified from the opisthobranchs including isonitriles (Hagedone et al., 1979;

Cimino et al., 1982; Walker, 1981), isothiocyanates and formamides (Walker,

1981); dials (Cimino et al., 1985a;b), trienones (Cimino et al., 1989), acid glycerides (Gustafson and Andersen, 1985) diacylglycerols (Cimino et al.,

1988), unusual fatty acids (Fenical et al., 1979), lactones (Ojika et al., 1990), pyrroles (Cartè and Faulkner, 1983; Paul et al., 1990) and macrolides (Pawlik et al., 1988). A wide range of nonhalogenated terpenes and furans are concentrated from sponges and other dietary sources (Walker, 1981; Faulkner,

1992).

The nudibranch Aldisa sanguinea cooperi modifies sterols to produce the bioactive metabolite 3-oxo-chol-4-ene-24-oic acid (Ayer and Anderson, 1982).

Polypropionate metabolites are produced by several sacoglossans (Ireland and

Scheuer, 1979; Ireland and Faulkner, 1981; Ksebati and Schmitz, 1985) and a number of pulmonates in the genus Siphonaria (Hochlowski et al., 1983a;

Manker et al., 1988; Manker and Faulkner, 1989a,b). Pulmonates have also been found to produce pyrones (Hochlowski et al., 1984) and diterpenes (Rice,

1985). A mixture of dietary sequestered aromatic metabolites have been isolated from prosobranch lamellariids (Andersen et al., 1985) and several species of Nerites produce antimicrobial isoflavones (Sanduja et al., 1985). Chapter 6: Volatile compounds in the egg masses of molluscs 261

Water soluble bioactive compounds from marine molluscs have been subject to less attention, although a wide range of species are known to produce defensive secretions composed primarily of sulphuric acid (Thompson, 1969).

Choline ester derivatives have been found in 55 species of mollusc in the superfamily muricoidea (Roseghini et al., 1996), as well as the Opisthobranch

Aplysia californica (Blankenship et al., 1975). Bioactive peptides have been isolated from Opisthobranchs (e.g. Bai et al., 1990), pulmomates (Fernández et al., 1996) and prosobranchs in the genus Conus (Olivera et al., 1990).

6.1.4 Metabolites from molluscan egg masses Only a small number of compounds have been identified from the egg masses of marine molluscs. The best studied are the egg ribbons of sea hares in the family Aplysiidae. An antineoplastic component has been isolated from the egg ribbons of Aplysia kurodai and characterised as a 250-kDa glycoprotein,

Aplysianin E (Yamazaki et al., 1985; Kisugi et al., 1987). Antigens against

Aplysianin E were found to recognise Julianin E, a cytolytic factor from the egg mass of Aplysia juliana (Yamazaki, 1993). The Julianins have a similar amino acid composition to the Aplysianins but differed in their sugar composition.

Antitumour activity was also reported in the eggs of another sea hare Dolabella auricularia but the active component(s) were not isolated (Yamazaki, 1993).

The cytolytic factor in the eggs of Dolabella auricularia was not recognised by antigens against the Aplysians or Julianins (Yamazaki, 1993), which suggests that biologically active glycoproteins could be conserved within the genus

Aplysia but diverge within the Aplysiidae family. Chapter 6: Volatile compounds in the egg masses of molluscs 262

Other metabolites isolated from the egg mass of Aplysia spp. include sterols and fatty acid derivatives. A series of neutral glycosphingolipids and one novel phosphonoglycosphingolipid have been isolated from the eggs of A. juliana

(Yamaguchi et al., 1992a;c). The fatty acid components of these glycolipids were primarily saturated palmitic (C16) and stearic (C18) acid, although a range of C16-C20 acids were found. One glycolipid was found to contain primarily 2- hydroxypalmitc acid (Yamaguchi et al., 1992c). Diacyl hexadecylglycerol (30), a lipid known to have laxative properties, was isolated from the egg mass of A. kurodai, along with a mixture of cholesteryl esters (31) and three nucleosides

(32-34) (Miyamoto et al., 1988a). A novel sterol, cholest-7-en-6R-methoxy-3S,

5R-diol (35) and cholest-7-en-3S,5R-triol (36) and were isolated from the eggs of A. juliana (Yamaguchi et al., 1992b). It is thought that these sterols could function as defence substances, since several biological activities have been reported for the 7,8-dihydroderivative (Yamaguchi et al., 1992b).

Bioactive metabolites isolated from the eggs of other opisthobranch molluscs include novel antitumor macrolides. Ulapualide A and B (37) were isolated from the egg mass of the nudibranch Hexabranchus sanguineus (Roesener and

Scheuer, 1986). Another novel macrolide with antifungal properties, Kabiramide

C (38), was isolated from the egg masses of an unidentified nudibranch

(Matsunaga et al., 1986). The saccoglossan Eylsia halimedae sequesters an aldehyde halimedatetraacetate (39) from a sponge and converts it to the corresponding alcohol (40) (Paul and Van Alstyne, 1988). This compound is Chapter 6: Volatile compounds in the egg masses of molluscs 263

OR' RO 30. 2,3-diacyl-1-O-hexadecylglycerol R, R’= a:b:c= 2:1:2

a O b O c O

RO 31. cholesteryl esters R = a:b:c= 24:15:1 a O b O c O

O OH NH 2 CH 3 N N HN N N

O N N N N N O O O HO HO HO

HO HO OH HO OH 32. 1-(2-deoxy-β-D- 33. adenosine 34. inosine ribofuranosyl)thymine

35. R=Me; Cholest-7-en-6R-methoxy-3S,5R-diol HO 36. R=H; Cholest-7-en-3S,5R,6R-triol OH OR Chapter 6: Volatile compounds in the egg masses of molluscs 264

OHC O N Me OAc R MeO O OMe N O O OH N

N O 37. Ulapalide A; R= O Ulapalide B; R=H, OCOCH(Ome)CH2OMe O

OHC O N Me OMe O MeO O Me N O O N OCONH 2

38. Karabirimide C N O

OH OMe

OAc R

AcO

OAc 39. R= CHO; Halimedatetraacetate OAc 40. R= CH2OH Chapter 6: Volatile compounds in the egg masses of molluscs 265

found in high concentrations in the egg mass of E. halimedae. The egg mass of the Opisthobranch Tethys fimbria was found to contain a complex mixture of prostaglandin 1,15-lactone fatty acid esters (Cimino et al., 1991a; Di Marzo et al., 1991). Similar compounds are found in the adult mollusc and these have been implicated in their defence mechanisms (Cimino et al., 1989; 1991b).

6.1.5 Chemical diversity and dereplication. Clearly, a large diversity of secondary metabolites are produced by marine molluscs and coupled with the large number of species that are yet to be examined this offers good potential for the discovery of novel biologically active compounds. Nevertheless, recent trends in marine natural products chemistry indicate that most bioactive extracts contain only known compounds (Faulkner,

1995). Consequently, it is necessary to develop rapid methods to avoid the duplication of earlier research. In a shipboard screening expedition, Rinehart et al. (1974; 1981) used gas chromatography/mass spectrometry (GC/MS) as the principle analytical method to assign structures. GC/MS analyses were carried out on all crude extracts of organisms with high antimicrobial activity or halogen content. Using this technique known compounds can be readily identified by their mass spectral fragmentation patterns and GC/MS behaviour, thus providing the opportunity to prevent duplication. Rinehart et al. (1974) identified over 100 new halogenated organic compounds in marine organisms by GC/MS, in addition to identifying several known compounds in different species.

Chapter 6: Volatile compounds in the egg masses of molluscs 266

6.2 Objectives

The primary objective of this component of the study was to determine if the same types of compounds were likely to be responsible for antimicrobial activity in the egg masses of different species of mollusc. In particular, three brominated indoles were found to be the principal active components in the egg masses of Dicathais orbita (Chapter 5) and these compounds could also be responsible for the observed antimicrobial activity in the egg masses of other muricids. Consequently, the egg masses from 23 molluscs, including six muricids were specifically examined for brominated indoles, using gas chromatography/mass spectroscopy. The volatile organic components of molluscan egg masses were also examined for the presence of other halogenated compounds and any potential antimicrobial agents were identified.

For the purpose of this study volatile is applied to those compounds that evaporate under temperatures of 290oC in the GC/MS. Only those compounds that are soluble in organic solvent (essentially DCM) have been examined.

Some of the primary metabolites found in molluscan egg masses were tested for antimicrobial activity using the FDA assay. Chapter 6: Volatile compounds in the egg masses of molluscs 267

6.3 Methods

6.3.1 Specimen collection and sample preparation The egg masses from 20 species of molluscs were collected along the Illawarra

Coast and the egg masses from a further three species were collected from the

Mediterranean Sea, Spain (Table 6.1). The sites of collection and the total amount of egg mass collected are listed in Chapter 4, Table 4.1. Most of the egg material was extracted immediately after collection. However, samples from some species were frozen or freeze-dried prior to extraction (refer to Table 6.1).

Organic extracts of the egg masses were prepared in chloroform/methanol, as described in Chapter 4, section 4.3.1. Two samples of the egg mass from

Conuber cf. sordidus were extracted in diethyl ether. Polar components were removed from the extracts by water/methanol separations with the chloroform or diethyl ether (refer to Chapter 4, section 4.3.1). These separations were carried out on extracts from fresh and frozen egg material but not on the extracts from freeze-dried samples.

Solvent controls were prepared by drying down 200ml of chloroform/methanol and diethyl ether. Environmental controls included chloroform/ methanol extracts of seawater, estuarine mud and intertidal pebbles. All control extracts were separated by silica chromatography in a similar manner to the egg extracts (refer to Chapter 4, section 4.3.1).

Chapter 6: Volatile compounds in the egg masses of molluscs 268

6.3.2 Derivatisation procedure Organic extracts from Aplysia spp. and Dicathais orbita were esterified to increase the volatility of the fatty acid components. The extracts (10 mg) were placed in 1 ml of 14% boron trifluoride (BF3) in methanol (w/v) in a 4 ml screw cap glass vial. The sample was sonicated and left at 800C for 30 mins. The vial was cooled and of distilled water (0.5 ml) was added. The mixture was shaken well and then hexane (1 ml) was added. The vial was vortexed before the hexane and water layers were allowed to separate. The hexane layer was removed with a Pasteur pipette and used for GC/MS analyses.

6.3.3 Gas chromatography/mass spectrometry analyses The volatile organic components in the egg masses were examined using a

GC-17A (Shimadzu) gas chromatograph coupled to a QP-5000 (Shimadzu) mass spectrometer. The samples were run in splitless mode, with the exception of the derivatised samples, which were run with a split ratio of 1:20. In general,

50 µl of sample dissolved in dichloromethane (DCM) was injected, at an estimated concentration of 10mg/ml. The samples were run using the parameters outlined in Chapter 5, Section 5.3.2. Three replicate runs were performed on the same extract from the egg masses of Dicathais orbita and

Aplysia juliana. Blank runs were performed sporadically to check for contaminants on the column or in the injection loop, by injecting 50 µl of DCM.

The retention time and intensity of each peak detected in the gas chromatograph was recorded for each sample. The composition of volatile organic compounds in the egg mass extracts was compared to the solvent Chapter 6: Volatile compounds in the egg masses of molluscs 269

controls and blank runs. Contaminants were removed and the relative intensity of each peak was calculated as a percent of the summed total.

Volatile compounds in the egg extracts were identified by their characteristic mass spectral fragmentation patterns. The fragmentation patterns were compared to known compounds contained in the mass spectrum library.

Monobrominated compounds were detected by the characteristic presence of paired peaks separated by 2 mass units and of equal intensity for the natural isotopes 79Br and 81Br. Similarly, dibrominated compounds were identified by the presence of triplets in the ratio of 1:2:1.

Extracts from the egg masses of Dicathais orbita, and a derivatised sample from Aplysia juliana, were also run on the GC/MS in the chemical ionisation mode using Helium as a carrier gas. This permitted the detection of the molecular ions of some of the volatile constituents in the egg masses. The samples were run in splitless mode using the same parameters outlined in

Chapter 5, Section 5.3.2.

6.3.4 Antimicrobial activity of identified egg constituents Several major constituents identified in the egg masses were tested for antimicrobial activity against Staphylococcus aureus, Escherichia coli and

Pseudomonas aeruginosa using the FDA assay (refer to Chapter 4). The compounds were obtained from Aldrich and include three fatty acids (stearic acid, heptanoic acid, oleic acid), a triglyceride (triolein), cholesterol and sulfur

(S8). All compounds were tested at concentrations of 1 and 0.1mg/ml. Chapter 6: Volatile compounds in the egg masses of molluscs 270

Table 6.1: Species of mollusc selected for GC/MS examination of volatile organic chemicals in the egg mass. The type of egg mass deposited by the species is noted (i.e. leathery egg capsules or gelatinous egg mass). The egg masses were either extracted immediately after collection (fresh) or after storage (frozen or freeze-dried). Derivatised samples were extracted fresh and then methylated. Number of samples refers to the number of extracts that were prepared from independent collections of the eggs.

Family Species Type of Method of No. egg mass preparation samples LITTORINIMORPHA Littorinidae Bembicium nanum Gelatinous Fresh 2 Naticidae Conuber c.f. sordidus Gelatinous Fresh 4 Freeze-dried 1 Ranellidae Cabestana spengleri Leathery Fresh 1 NEOGASTROPODA Muricidae Agnewia tritoniformis Leathery Fresh 2 Dicathais orbita Leathery Fresh 3 Frozen 1 Freeze-dried 2 Derivatised 1 Hatching 1 Ceratostoma erinaceum* Leathery Freeze-dried 1 Lepsiella reticularis Leathery Fresh 1 Morula marginalba Leathery Fresh 2 Trunculariopsis trunculus* Leathery Freeze-dried 1 Mitridae Mitra carbonaria Leathery Fresh 1 Conidae Conus paperliferus Leathery Fresh 1 PULMONATA Amphibolidae Salinator fragilis Gelatinous Frozen 2 Salinator solida Gelatinous Frozen 1 Siphonariidae Siphonaria denticulata Gelatinous Fresh 2 Siphonaria zelandica Gelatinous Fresh 1 Planorbidae Isidorella hainesi Gelatinous Fresh 1 OPISTHOBRANCHIA Aplysiidae Aplysia sp. Gelatinous Freeze-dried 1 Aplysia juliana Gelatinous Frozen 1 Derivatised 1 Aplysia parvula Gelatinous Derivatised 1 Aplysia sydneyensis Gelatinous Derivatised 1 Stylocheilus longicauda Gelatinous Fresh 1 Philinidae Philine angasi Gelatinous Frozen 1 Glaucidae Spurilla neopolitana* Gelatinous Freeze-dried 1 CEPHALOPODA Loliginidae Sepioteuthis australis Tough Fresh 1 capsules Freeze-dried 1 * Obtained from the Mediterranean Sea, Spain. All other specimens were collected along the Illawarra Coast, N.S.W. Australia (see Chapter 4, Table 4.1). Chapter 6: Volatile organic compounds in molluscan egg masses 271

6.4 Results

6.4.1 Brominated compounds in Muricid egg masses The egg masses from all six species of Muricidae were found to contain brominated indoles (Table 6.2; Appendix 6.1a-f). However, this type of compound was not found in extracts from any other molluscs, including the neogastropods, Mitra carbonaria and Conus paperliferus (Appendix 6.1).

Although, it should be noted that the fresh egg capsules of Mitra carbonaria rapidly changed colour on exposure to air. The extract from these egg capsules was also highly insoluble and overall, few volatile compounds were found in this extract (Appendix 6.1g).

The total number of brominated compounds found in the egg masses of the

Muricidae was 23 (Table 6.2; Appendix 6.2). The number of brominated compounds found in a single extract varied from one in Agnewia tritoniformis to a maximum of twelve in Trunculariopsis trunculus (Table 6.2). The number of brominated compounds found in the egg mass of Dicathais orbita, varied from 3 to 11 in the different extracts (Appendix 6.1c). Overall, a total of 14 brominated compounds was found in the egg mass of D. orbita (Table 6.2). The molecular ions of several brominated compounds in Table 6.2 have been assigned based on an extract from the eggs of D. orbita that was run in chemical ionization mode on the gc/ms (refer to Appendix 6.3). Eight brominated compounds were detected on this occasion.

The proportion of brominated metabolites found in the Muricid egg masses also varied between species. Brominated metabolites constitute more than 28% the Chapter 6: Volatile organic compounds in molluscan egg masses 272

volatile organic compounds in the fresh egg mass of Dicathais orbita (Table

6.3). A reasonably high proportion of brominated compounds were also found in the extracts from the egg masses of the Australian muricids, Lepsiella reticularis and Morula marginalba, as well as the Mediterranean species Ceratostoma erineceum (Table 6.3). Brominated metabolites formed less than 10 per cent of the volatile organic extract from the egg mass of Trunculariopisis trunculus

(Table 6.3). Notably, the extract from this species contained a large proportion of totally insoluble material. Less than one per cent of the extract from the eggs of Agnewia tritoniformis was represented by brominated metabolites (Table

6.3). However, the eggs of Agnewia tritoniformis rapidly turned purple during the extraction process. The hatching (purple) eggs of Dicathais orbita were not found to contain any brominated compounds and no brominated compounds were found in a derivatised extract from the fresh egg mass of D. orbita (Table

6.3; Appendix 6.1c)

The majority of brominated compounds found in the Muricid egg masses were monobrominated (Table 6.2). Based on the mass spectral fragmentation patterns seven dibrominated compounds and two tribrominated compounds were also found (Appendix 6.2). Both of the tribrominated compounds and all but one of the dibrominated compounds were exclusively found in the two

Mediterranean Muricids (Table 6.2). In total, eleven brominated metabolites were unique to one species of Muricid (Table 6.2).

Chapter 6: Volatile organic compounds in molluscan egg masses 273

Table 6.2: Volatile brominated compounds in extracts from the egg masses of four Australian and two Mediterranean Muricid molluscs. The species are; A.t.- Agnewia tritoniformis; D.o.- Dicathais orbita; L.r.- Lepsiella reticularis; M.m.- Morula marginalba; C.e.- Ceratostoma erineceum; T.t.- Trunculariopsis trunculus. R.T. refers to retention time (mins) in the gas chromatographer. Major ions refers to brominated fragment ions in the mass spectrometer of >20% relative abundance. MI = Molecular ion. The symbol + indicates that the compound is present in that particular species.

R.T. Identity Major ions Australian species Mediterranean (Br=79) A.t. D.o. L.r. M.m. C.e. T.t.

18.5 Di brominated 174, 217 + 27.8 44/45 Dibromo 199, MI 226 + + pyrazole/imidazole 29.0 44/45 Dibromo 199, MI 226 + pyrazole/imidazole 30.3 Brominated indole 196 + 30.6 Di brominated 239, 277, 318 + 30.9 Di brominated 233, 260 + 32.2 42 6-Bromo-2-methoxy- 224, MI 239 + + + 3H- indol-3-one 32.4 Brominated indole 197, MI 229 + + + + 33.4 46/47 Tribromo 277, MI 304 + + pryazole/imidazole 33.6 46/47 Tribromo 277, MI 304 + + pyrazole/imidazole 34.5 Brominated indole 224, MI 253 + + 34.6 43 4-bromoanthranilic 197, MI 215 + acid 36.1 Brominated indole 224, MI 255 + 37.5 14 6-Bromoindoxyl 182, MI 211 + 38.0 3 Tyrindoleninone 240, MI 255 + + + + + + 38.8 Dibrominated compound 58, 296 + 39.1 Brominated indole 225, 257, 297 + + 39.3 Brominated indole 223, MI 252 + 41.6 41 6-Bromo-2- 224, MI 271 + + methylsulfinyl-3-indolone 42.5 7 6-Bromoisatin 170, 197, 225 + + + + + 43.1 Brominated indole 213, 241, 269 + + 45.0 4 Tyriverdin 210, 242, 257 + + + + + 47.0 Brominated compound 165, 206 + Number of brominated compounds 23 2 14 4 6 10 12 Chapter 6: Volatile organic compounds in molluscan egg masses 274

Table 6.3: The composition of brominated compounds in the egg masses of 6 Muricid molluscs. The extracts have been taken from fresh eggs unless otherwise stated. Fz refers to extracts from frozen eggs and FD refers to extracts from freeze-dried egg material. The percent of all brominated compounds in the extracts is estimated from the relative intensities of all volatile organic constituents in the gas chromatograph. The maximum number of brominated compounds found in the extracts from each species is also listed. The proportion of four brominated metabolites related to Tyrian Purple is expressed as the percent of all volatile brominated compounds found in the extract. These brominated metabolites are; 3 = tyrindoleninone; 4 = tyriverdin; 7 = 6-bromoisatin; and 41= 6-bromo2-methylsulfinyl-3-indolone. Others refers to the combined intensity of all brominated metabolites other than these four compounds. Where duplicate extracts were run the values are expressed as a mean with the standard error in parentheses.

Species % of volatile No. Br Percent of Br compounds compounds compounds 3 4 7 41 Others A. tritoniformis 0.5 1 100 0 0 0 0 C. erineceum FD 16.4 11 5.3 5.7 19.9 18.3 57.0 D. orbita 31.9 (5.1) 10 74.1 0.4 13.4 2.6 9.5 (3.5) (0.4) (8.4) (2.6) (6.5) D. orbita Fz 36.9 7 44.8 0.8 6.2 31.6 9.1 D. orbita FD 28.75 (3.2) 11 38.5 1.95 11.2 35.4 16.2 (3.5) (2.0) (6.7) (4.9) (9.5) D. orbita Derivatised 0 0 0 0 0 0 0 D. orbita Hatching 0 0 0 0 0 0 0 L. reticularis 24.4 2 97.2 0 2.9 0 0 M. marginalba 15.9 (2.7) 5 24.8 25.3 18.7 0 31.3 (10.5) (25.3) (11.1) (3.7) T. trunculus FD 7.2 12 2.3 11.4 1.4 3.2 91.9

Chapter 6: Volatile organic compounds in molluscan egg masses 275

Only two brominated compounds could be positively identified from the mass spectrum library, 6-bromo-2-methylthio-3H-indol-3-one (Tyrindoleninone 3;

Appendix 6.2a) and 6-bromoindole-2,3- dione (bromoisatin 7; Appendix 6.2b).

This compound is assumed to be the 6-bromo rather than the 5-bromo isomer

(refer to Chapter 5). A dibrominated compound with a retention time of 49-52 mins also showed a positive match to tyrindoleninone (Appendix 6.2c). This compound did not produce a sharp peak in the GC and is thought to be a decomposition product of the unstable and nonvolatile compound 2,2’- bis(methylthio)-6,6’-dibromoindigotin (tyriverdin 4). The structures of three further brominated indoles have been assigned tentatively on the basis of the mass spectral fragmentation patterns (refer to appendix 6.2d-f) and by comparison to the known compounds 3 and 4. These are 6-bromoindoxyl (14),

6-bromo-2-methylsulfinyl-3H- indol-3-one (41), and 6-bromo-2-methoxy-3H- indol-3-one (42). Similarly one compound has been tentatively identified as 4- bromoanthranilic acid (43; Appendix 6.2g).

Tyrindoleninone (3), was found in the egg masses of all of the muricids that were examined (Table 6.2). This was the major brominated component in extracts from the fresh eggs of the Australian muricids Dicathais orbita,

Agnewia tritoniformis and Lepsiella reticularis (Table 6.3). A significant proportion of tyrindoleninone was also found in the frozen and freeze-dried egg mass of Dicathais orbita, as well as the fresh eggs of Morula marginalba. By comparison, only a small proportion of the brominated compounds was represented by tryindoleninone in the freeze-dried egg masses of the Chapter 6: Volatile organic compounds in molluscan egg masses 276

Mediterranean muricids, Trunculariopsis trunculus and Ceratostoma erineceum

(Table 6.3).

The proportion of 6-bromo-2-methylsulfinyl-3H-indol-3-one (41) was found to increase relative to the proportion of tyrindoleninone (3) in the freeze-dried and frozen eggs of Dicathais orbita (Table 6.2). This compound was also found in extracts from the freeze-dried eggs of Trunculariopsis trunculus and

Ceratostoma erineceum but not in extracts from the fresh eggs of any other muricid (Table 6.3). The freeze-dried extracts from D. orbita egg capsules also produced an insoluble red compound, which did not pass through the silica column. A similar red substance was observed in relatively large quantities in the extract from T. trunculus egg mass. Extracts from all muricid egg masses contained some insoluble purple material that did not pass through the silica columns.

6-Bromoisatin (7), a decomposition product from the oxidation of tyriverdin (4), was found in the egg masses of all the muricids examined, with the exception of

Agnewia tritoniformis (Table 6.2). The compound most likely to be tyriverdin (4) was also found in most species (Table 6.2). The relative proportion of 6- bromoisatin (7) and tyriverdin (4) varied substantially in two different extracts from the eggs of Morula marginalba. In the two Mediterranean species the overall proportion of the four Tyrian Purple related compounds was low in comparison to other brominated metabolites.

Chapter 6: Volatile organic compounds in molluscan egg masses 277

O O

SCH3 O N N Br Br H

3. Tyrindoleninone 7. 6-Bromoisatin

O H Br SCH3 N

N Br H H 3CS O

4. Tyriverdin

OH O

O-

S CH + 3 N Br N Br

14. 6-bromoindoxyl 41. 6-bromo-2-methylsulfinyl- 3H-indol-3-one

O O

O- OCH 3 N + Br Br NH3

42. 6-bromo-2-methoxy- 43. 4-bromoanthranilic acid 3H-indol-3-one Chapter 6: Volatile organic compounds in molluscan egg masses 278

Two further compounds from the Mediterranean muricids gave positive matches to the library spectrum of a dibromo-1H-pyrazole (44) or a dibromo-1H- imidazole (45) (Table 6.3; Appendix 6.2h,i). However, based purely on the fragmentation patterns, it is not possible to determine which of these two types of compounds are present in the egg masses or the bromo substitution pattern(s). Similarly, two compounds with different gc retention times were found in the muricid egg masses, which showed positive matches to the mass spectrum of tribromoimidazole (46) and tribromopyrazole(47) (Appendix 6.2j,k).

The two compounds were not found together in the extracts from one species but both were minor metabolites in one Australian and one Mediterranean species (Table 6.2).

6.4.2 Other related compounds from muricid egg masses. Several nonbrominated indole derivatives were also found as minor metabolites in the egg masses of the two Mediterranean muricids (Appendix 6.1c,f). Four of these compounds gave positive matches in the mass spectrum library to 2- methylthio-3H-indol-3-one (20), 1H-Indole-2,3-dione (48), 1H-isoindole-1,3 (2H)- dione (49) and 2-aminobenzoic acid methyl ester (50) (refer to Appendix 6.4a- d). 2-Methylthio-3-indolone (20) was present in both Trunculariopsis trunculus and Ceratostoma erineaceum, whereas isoindole-1,3-dione (49) was only present in C. erineaceum. Indole-2,3-dione (48), as well as 2-aminobenzoic acid methyl ester (50) were only present in T. trunculus. The presence of 2- methylindene-1,3-dione (51) in the egg mass of both Mediterranean muricids has also been inferred from the mass spectral fragmentation pattern (refer to

Appendix 6.4e). Chapter 6: Volatile organic compounds in molluscan egg masses 279

H H H N N N N N N Br Br

Br Br Br Br

44. Dibromo-1H-pyrazoles

Br H H Br H N N N Br Br N N N Br Br

45. Dibromo-1H-imidazoles

H N Br H N N Br Br Br N Br Br

46. 3,4,5-Tribromo-1H-pyrazole 47. 2,4,5-Tribromoimidazole Chapter 6: Volatile organic compounds in molluscan egg masses 280

Two unidentified compounds found in the egg masses of the Mediterranean

Muricids are also likely to be indole derivatives based on their mass spectral fragmentation patterns (Appendix 6.4f,g). One compound showing a similar fragmentation pattern to 3-methyl-5-phenyl-1H-pyrazole (52) was found in the egg mass of Ceratostoma erineceum (Appendix 6.4h).

The egg mass of Trunculariopsis trunculus was also found to contain the volatile compound methyl methanethiosulfonate (53) (Appendix 6.5a). An unidentified sulfoxide was also detected in the eggs of both T. trunculus and

Ceratostoma erineceum (Appendix 6.5b). Dimethyltrisulfide (54) was found in egg mass of C. erineceum (Appendix 6.5c) and an unidentified disulfide was detected in the egg mass of Dicathais orbita (Appendix 6.5d).

6.4.3 Halogenated compounds from Aplysiidae egg masses Members of the family Aplysiidae were the only other molluscs found to contain halogenated compounds in their egg masses. These were all small chlorinated compounds that were minor metabolites, constituting a small proportion of all volatile organic constituents (Table 6.4). Halogenated compounds were found in the egg masses of two species in the family Aplysiidae, Aplysia sp. and

Stylocheilus longicauda (Appendix 6.1i,m). The egg ribbons of Aplysia sp. that were found to contain halogenated metabolites were freeze-dried immediately after collection and then extracted and analysed without lengthy periods of storage. No halogenated compounds were found in extracts from Aplysia juliana egg ribbons that had been stored in the freezer or in the derivatised samples Chapter 6: Volatile organic compounds in molluscan egg masses 281

O O

SCH3 OH N N

20. 2-methylthio-3H-indol-3-one 48 1H-indole-2,3-dione

O O

CH3 O NH

NH2 O

49. 1H-isoindole-1,3 (2H)-dione 50. 2-aminobenzoic acid methyl ester

O HN

CH3

51. 2-methylindene-1,3-dione 52. 3-methyl-5-phenyl-1H-pyrazole

S S S S S O

53. methyl methanethiosulfonate 54. dimethyltrisulfide Chapter 6: Volatile organic compounds in molluscan egg masses 282

from three species of Aplysia (Table 6.4; Appendix 6.1j-l). The extract from

Stylocheilus longicauda that was found to contain halogenated compounds was taken from fresh eggs immediately after collection.

A total of 32 halogenated compounds were found in the egg ribbons of Aplysia sp. (Table 6.4). A range of small mono-, di-, tri and polychlorinated compounds were found (Appendix 6.6). Only six of the chlorinated compounds showed positive matches to compounds in the mass spectrum library. These were methyl trichloroacetate (55), 1,1,3-trichloropropanone (56), 2-chloro-1-propene

(57), 2,2-dichloro-methylpropanal (58), 3-chloro-2-(chloromethyl)-1-propene

(59) and 1,3-dichloropropanone (60) (Appendix 6.6a-f). Two further compounds have been identified as isomers of trichloropentene (e.g. 61) and tetrachloropentane (e.g. 62) based on the fragmentation pattern (Appendix 6.6g

&h).

The egg ribbons of Stylocheilus longicauda were only found to contain five chlorinated compounds (Table 6.4). Two of the chlorinated compounds were the same as those found in the eggs of Aplysia juliana and these are tetrachloropentane (e.g. 62) and one unidentified compound (Appendix 6.6l).

Benzylchloride (63) and benzyl chlorformate (64) have been positively identified in the eggs of S. longicauda (Appendix 6.6 i&j). A compound with a similar fragmentation pattern to 2-phenoxypropanoyl chloride was also found

(Appendix 6.6k). Chapter 6: Volatile organic compounds in molluscan egg masses 283

Cl Cl O

Cl Cl Cl Cl O O

55. methyl trichloroacetate 56. 1,1,3-trichloropropanone

Cl O Cl

Cl H

57. 2-chloro-1-propene 58. 2,3-dichloro-2-methylpropanal

Cl Cl Cl Cl

59. 3-chloro-2-(chloromethyl)-1-propene 60. 1,3-dichloropropanone

Cl Cl

Cl Cl Cl Cl Cl

61. 1,1,5-tricholoro-2-pentene 62. 1,2,3,5-tetrachloropentane

Cl O

O Cl

63. Benzylchloride 64. Benzyl chloroformate Chapter 6: Volatile organic compounds in molluscan egg masses 284

Table 6.4: The composition of chlorinated compounds in Aplysiidae egg masses. The percent of chlorinated compounds found in the extracts is estimated from sum of the relative intensities of all volatile organic constituents in the gas chromatograph. The number of chlorinated compounds found in the extract is recorded for each species.

Species % of total Number of compounds

Aplysia sp. Freeze dried 14.4 32

Aplysia juliana Frozen 0 0

Aplysia juliana Derivatised 0 0

Aplysia parvula Derivatised 0 0

Aplysia sydneyensis Derivatised 0 0

Stylocheilus longicauda Fresh 3.9 6

Chapter 6: Volatile organic compounds in molluscan egg masses 285

6.4.4 Fatty acids and methyl esters Fatty acids were found in extracts from all the molluscan egg masses that were examined, with the exception of Mitra carbonaria (Appendix 6.1). For the purpose of this study all fatty acids and the methyl esters of fatty acids have been combined. This is because some of the samples have been methylated for analysis. Furthermore, the egg extracts were prepared in methanol and therefore the fatty acids could have been methylated during storage due to traces of methanol remaining in the samples. Consequently, it is not possible to determine whether the methyl esters are real components of the egg extracts or artefacts from the methylation of naturally occurring fatty acids.

There is considerable variation in the amount of fatty acid components found in extracts from the egg masses of marine molluscs (Table 6.5; 6.6). In general, fatty acids were found to be minor components in the leathery egg capsules and by comparison, a high proportion of fatty acids was found in gelatinous molluscan egg masses (Figure 6.1a). In particular, extracts from the gelatinous egg masses of species in the Aplysiidae family were dominated by long chain

(C18-C22) saturated and unsaturated fatty acids (Table 6.5; 6.6; Appendix 6.1i- m).

The fatty acid composition was found to vary significantly between the major taxonomic groupings of marine molluscs (Figure 6.1b). However, the composition of fatty acids also varies within each of these taxonomic groupings

(Table 6.5; 6.6). The least variation in fatty acid composition was observed in the Neogastropods, which were found to contain a relatively small proportion of Chapter 6: Volatile organic compounds in molluscan egg masses 286

exclusively saturated fatty acids (Figure 6.1b). The leathery egg capsules of the

Littorinimorpha Cabestana spengleri were similar to the neogastropods, with no unsaturated acids and a small overall proportion of fatty acids (Table 6.5; 6.6).

The egg masses from species in every other taxonomic group contained a mix of saturated and unsaturated fatty acids (Figure 6.1b).

The major saturated fatty acid found in all molluscan egg masses was palmitic acid (C16) (Table 6.5). Palmitic acid was the only acid found in the egg capsules of two neogastropods (Table 6.5). Stearic acid (C18) was also common in the molluscan egg masses and tetradecanoic acid (C14) was found in most of the gelatinous egg masses (Table 6.5). Fatty acids with an odd number of carbon atoms were uncommon in the neogastropod egg capsules. A high proportion of heptadecanoic acid distinguished the leathery egg capsules of the Littorinimorpha Cabestana spengleri from the neogastropods egg capsules (Table 6.5). A particularly unusual fatty acid composition was found in the egg ribbons of the nudibranch Spurilla neopolitana. The saturated fatty acids of this species were dominated by pentadecanoic (C15) acid (Table 6.5).

A compound with a similar mass spectrum fragmentation pattern to octadecanoic acid oxy methyl ester was also identified in S. neopolitana egg ribbons (Appendix 6.1n).

Most of the unsaturated fatty acids found in the molluscan egg masses have an even chain length (Table 6.6). Oleic (C18 enoic acid) was the dominant unsaturated fatty acid, although C16 and C20 unsaturated acids were also common (Table 6.6). Opisthobranchs in the genus Aplysia were found to Chapter 6: Volatile organic compounds in molluscan egg masses 287

contain a large number of polyunsaturated C20 acids, including dieneoic, trienoic, tetraenoic and pentenoic acids (Appendix 6.1i-m). Polyunsaturated acids with carbon chains of 18 and 22 were also found in the gelatinous egg mass of Aplysia spp. and Bembicium nanum (Table 6.5; Appendix 6.1i-m,u).

Extracts from the egg ribbons of S. longicauda and Aplysia juliana were found to contain some unidentified unsaturated acids, which could be C21 eneoic and

C21 trienoic acid (Table 6.5; Appendix 6.1j,m). These acids did not produce a molecular ion in the mass spectrometer and their retention time did not match any of the C20 or C22 acids, but fell in between these two groups of acids.

Overall, a higher proportion and a greater range of unsaturated fatty acids were identified in Aplysia extracts that were methylated (derivatised) prior to being analysed on the gc/ms (Table 6.5).

6.4.5 Other volatile components of molluscan egg masses Overall, a large diversity of volatile organic compounds was found in the egg masses of marine molluscs. The extracts from each species was found to produce a unique profile in the gas chromatograph with a range of unique compounds present and with different proportions of the common compounds

(refer to Appendix 6.1). The extracts of most species contained a large number of minor metabolites, which could not be identified readily. The chemical composition of the molluscan extracts was reproducible in replicate runs of the same extract. However, considerable intraspecies variation occurred in extracts that were prepared from different collections of the same species. Chapter 6: Volatile organic compounds in molluscan egg masses 288

Saturated 30 Unsaturated N = 21

25 a) 20

15 N = 14

10 % volatile organic compounds

0 Leathery capsules Gelatinous masses

40 N= 2

35 N= 8 30 N= 8 N= 7 b) Saturated 25 Unsaturated

15 N= 13

% volatile organic compounds 10

0 Opisthobranchia Pulmonata Littorinimorpha Neogastropoda Cephalopoda

Figure 6.1: A comparison of composition of saturated and unsaturated fatty acids and the methyl esters of fatty acids found in; a) the leathery egg capsules and gelatinous egg masses deposited by molluscs and; b) the egg masses of molluscs from five major taxonomic groups. The proportion of fatty acids/methyl esters is expressed as the mean percent of all the volatile organic compounds found in extracts from a number of species in each taxonomic group. N refers to the total number of extracts that were run in each group. Chapter 6: Volatile organic compounds in molluscan egg masses 289

Table 6.5: The composition of saturated fatty acids/ methyl esters in the egg masses of 23 species of molluscs from 5 taxonomic groups. The overall proportion of the volatile organic compounds represented by saturated fatty acids is listed. The percent of saturated fatty acids represented by each of the primary fatty acids found in the egg extracts is also recorded.

TAXON Proportion Percent of saturated acids Species of volatile C14 C15 C16 C17 C18 C20 Other compounds s LITTORINIMORPHA Bembicium nanum 1 47.8 23.0 0 50.6 2.9 9.0 13.4 1.0 Bembicium nanum 2 43.4 16.1 3.7 30.4 17.3 27.4 3.4 1.6 Conuber sordidus 1 1.6 0 0 87.5 0 0 0 12.5 Conuber sordidus 2 19.2 16.7 7.3 31.8 13.5 7.3 0 23.3 Conuber sordidus 3 42.7 67.2 2.6 17.1 3.3 9.8 0 0 Conuber sordidus 4 36.7 21.8 4.6 53.9 9.4 10.2 0 3.3 Conuber sordidus FD 14.5 69.0 0 24.8 6.2 0 0 0 Cabestana spengleri 6.5 0 0 13.8 33.8 52.3 0 0 NEOGASTROPODA Agnewia tritoniformis 1.9 0 0 100 0 0 0 0 Ceratostoma erineceum FD 5 0 0 0 0 0 0 5 Dicathais orbita 1 1.4 0 0 100 0 0 0 0 Dicathais orbita 2 10 0 0 48.5 0 44.6 0 1.4 Dicathais orbita Frozen 2.9 0 0 100 0 0 0 0 Dicathais orbita FD 1 13.2 0 0 57.1 12.8 24.1 0 6.0 Dicathais orbita FD 2 11.3 0 0 100 0 0 0 0 Lepsiella reticularis 21 0 0 61.0 0 39.0 0 0 Morula marginalba 1 27.2 0 0 61.8 0 38.2 0 0 Morula marginalba 2 25.2 0 0 67.9 0 32.1 0 0 Trunculariopsis trunculus FD 4.6 13.0 0 23.9 8.7 30.4 0 23.9 Conus paperliferus 2.6 0 0 100 0 0 0 0 Mitra carbonaria 0 0 0 0 0 0 0 0 PULMONATA Siphonaria denticulata 1 13.4 14.9 5.2 53.7 11.2 13.4 0 1.5 Siphonaria denticulata 2 22.6 10.6 4.0 47.8 6.2 22.1 4 5.3 Siphonaria zelandica 28.4 9.9 2.5 38.7 12.3 19.7 0 16.9 Salinator fragilis 1 33.9 0 0 100 0 0 0 0 Salinator fragilis 2 11 8.2 6.4 77.3 0 8.2 0 0 Salinator solida 26.6 20.7 12.4 53.4 10.9 2.6 0 1.5 Isidorella hainesi 4.6 0 6.5 67.4 0 26.1 0 0 OPISTHOBRANCHIA Aplysia sp. FD 24.3 2.4 3.3 9.9 48.9 34.9 0 0 Aplysia juliana 26.8 6.7 2.6 29.9 23.1 34.7 0 3.0 Aplysia juliana Derivatised 27.5 9.8 4.4 41.5 18.2 23.6 1.5 1.1 Aplysia parvula Derivatised 16.5 7.3 3.6 32.7 33.3 21.8 0 1.2 A. sydneyensis Derivatised 44.6 7.0 3.6 41.3 20.2 28.0 0 0 Stylocheilus longicauda 31.4 9.2 4.1 37.6 20.4 21.7 0 6.2 Philine angasi 28.4 13.7 8.5 45.4 10.6 19.7 0 2.1 Spurilla neopolitana Freeze D. 6.8 0.1 93.1 3.0 0 1.5 1.5 0 CEPHALOPODA Sepioteuthis australis 1 30.7 17.3 2.6 69.0 0 11.1 0 0.3 Sepioteuthis australis 2 40.7 13.3 2.9 45.5 7.4 31.0 0 0

Chapter 6: Volatile organic compounds in molluscan egg masses 290

Table 6.6: The composition of unsaturated fatty acids/ methyl esters in the egg masses of 15 species of molluscs from 4 taxonomic groups. The overall proportion of volatile organic compounds represented by unsaturated fatty acids is listed. The percent of unsaturated fatty acids is recorded for each of the primary unsaturated acids found in the egg extracts.

TAXON Proportion of all volatile Percent of unsaturated acids Species compounds C18 C16 C18 C20 C22 Others LITTORINOMORPHA Bembicium nanum 1 27.3 6.2 56.1 31.5 6.2 0 Bembicium nanum 2 19.2 29.2 29.7 37.5 0 3.6 Conuber sordidus 1 0 0 0 0 0 0 Conuber sordidus 2 3.9 0 74.4 0 25.6 0 Conuber sordidus 3 1.4 0 100 0 0 0 Conuber sordidus 4 12.9 44.2 43.4 6.9 0 5.4 Conuber sordidus Freeze d. 6 0 100 0 0 0 Cabestana spengleri 0 0 0 0 0 0 PULMONATA Siphonaria denticulata 1 3.4 20.6 79.4 0 0 0 Siphonaria denticulata 2 9.2 6.5 57.6 18.5 0 17.4 Siphonaria zelandica 6.7 0 100 0 0 0 Salinator fragilis 1 17.2 100 0 0 0 0 Salinator fragilis 2 16.9 63.9 29.0 1.8 0 5.3 Salinator solida 41.2 36.4 45.1 6.8 0 11.7 Isidorella hainesi 1.6 0 100 0 0 0 OPISTHOBRANCHIA Aplysia sp. Freeze dried 18.6 4.8 44.1 16.7 35.5 0 Aplysia juiliana 14.7 7.5 19.7 26.5 2.7 43.5 Aplysia juliana Derivatised 46.9 4.5 25.6 47.3 19.0 3.6 Aplysia parvula Derivatised 27.6 2.9 26.8 55.1 15.2 0 Aplysia sydneyensis Deriv. 47.4 0 45.1 44.5 10.3 0 Stylocheilus longicauda 18.5 17.3 51.9 0 25.9 4.9 Philine angasi 5.8 18.1 49.7 31.0 1.7 0 Spurilla neopolitana F.D. 5.3 0 0 2.8 0 96.2 FFrreeze D.Freeze CEPHALOPODA Sepioteuthis australis 1 1.7 100 0 0 0 0 Sepioteuthis australis 2 7.9 32.89 67.1 0 0 0

Chapter 6: Volatile organic compounds in the egg masses of molluscs 291

The organic extracts from the egg masses of al molluscs examined were found to contain a significant proportion of sterols (Figure 6.2). Overall, the neogastropods were found to contain the highest proportion of sterols and the opisthobranchs contained the lowest. However, the amount of sterols found in the individual species from each taxonomic group did vary substantially

(Appendix 6.1). Cholesterol was the only sterol found in the egg mass of every species that was examined and was the most abundant sterol in every egg mass (Table 6.7; Appendix 6.1).

Sulfur was present in relatively high concentrations in extracts from the egg ribbons of Salinator fragilis (Table 6.7). Both the S6 (65) and S8 (66) forms of sulfur were positively matched on the mass spectrum library (Appendix 6.7a,b).

Hexathiepane (67) was also identified from the S. fragilis egg extract (Appendix

6.7c). Two independent extracts from S. fragilis egg ribbons were analysed. The first extract was analysed immediately after collection, whereas the second extract was analysed approximately six weeks after preparation. The first extract was also run at a lower concentration (~1mg/ml). The concentration of the soluble (S6) form of sulfur was significantly lower in the older extract although the concentration of S8 remained fairly high (Table 6.7). Hexathiepane was only found in the second extract, which was run at a higher concentration

(~10mg/ml). The two sulfur molecules and hexathiepane were also found in extracts from the estuarine mud (Table 6.7, Appendix 6.8a). This mud was collected from the same site as the egg ribbons of Salinator fragilis. Sulfur was also found in extracts taken from intertidal pebbles (Table 6.7, Appendix 6.8b) but was not in the eggs of any intertidal reef molluscs (refer to Appendix 6.1). Chapter 6: Volatile organic compounds in the egg masses of molluscs 292

40 N = 2

35 N = 13 other sterols 30 cholesterol

25 N = 8

20 N = 8 N = 7

% volatile organic compounds 10

0 Opisthobranchia Pulmonata Littorinimorpha Neogastropoda Cephalopoda

Figure 6.2: The mean proportion of cholesterol and other sterols found in organic extracts from the egg masses of 23 molluscs in five major taxonomic groups. Standard deviations are provided for the total sterol content and N refers to the number of extracts that were analysed in each taxonomic group. Chapter 6: Volatile organic compounds in the egg masses of molluscs 293

Table 6.7: The relative abundance of sulfur and hexathiepane in extracts from the egg ribbons of the estuarine snail Salinator fragilis and two intertidal substrates.

Sample % volatile organic compounds

S6 S8 Hexathiepane

Salinator fragilis 1 8.1 18.1 0

Salinator fragilis 2 0.3 10.4 0.2

Estuarine mud 0.3 12.5 0.3

Intertidal pebbles 0 6.1 0

s s s s s s s

65. Sulfur (S6) 66. Sulfur (S8)

s s

s s s s

67. Hexathiepane Chapter 6: Volatile organic compounds in the egg masses of molluscs 294

The egg masses of the pulmonates and opisthobranchs were found to contain a large amount of unidentified unsaturated material (Appendix 6.1). Most of these compounds produced mass spectral fragmentation patterns that were similar to aldehydes, ketone and alcohols in the mass spectrum library. Several compounds showing positive library matches to the ethyl esters of fatty acids were consistently found in extracts taken from the gelatinous egg mass of

Conuber sordidus (Appendix 6.1t). The ester isopropyl myristate was found in the egg extracts from many molluscs, including both Australian and

Mediterranean species. This compound was the major volatile organic compound found in an extract taken from seawater (Appendix 6.8c).

Phthalates and alkanes were the dominant components of the chlorinated solvent controls (Appendix 6.8d-f). Many of these compounds were also found in the egg extracts but these were removed for the purpose of analysis.

A large number of unidentified compounds, many which appeared to be ketones, were also found in the solvent controls (Appendix 6.9). Only a few of these matched components found in the egg extracts. However, it is likely that some of the minor metabolites found in the egg extracts are solvent contaminants because controls were not prepared from every batch of solvent that was used. Different solvent contaminants could be partly responsible for some of the intraspecies variation that was observed in the extracts from molluscan egg masses.

A solvent control was also prepared from diethyl ether, which was used to extract the eggs of Conuber sordidus on one occasion. The diethyl ether was found to contain phthalates and a range of phenolic compounds including Chapter 6: Volatile organic compounds in the egg masses of molluscs 295 butylhydroxytoluene (Appendix 6.8g). These phenols were also found in the C. sordidus extract. A range of phenolic and other aromatic compounds were also found in an extract from the eggs of Aplysia parvula (Appendix 6.1k). This extract was not prepared in ether and the aromatic compounds were not found in any solvent controls or the extracts from other species (refer to Appendix 6.1;

6.8).

6.4.6 Antimicrobial activity of some common egg metabolites Some of the common compounds found in the egg masses or marine molluscs were tested for antimicrobial activity (Table 6.8). The compounds were tested at concentrations above that expected to occur in the egg masses. The unsaturated fatty acid, oleic acid, was the only compound found to exhibit significant antimicrobial activity (Table 6.8). This compound was found to completely inhibit the hydrolysis of fluorescein diacetate at 10mg/ml and was partially active at 1mg/ml. Two saturated fatty acids, stearic and heptadecanoic acid, as well as the triglyceride triolein, showed partial activity at a concentration of 10 mg/ml (Table 6.8). Cholesterol and sulfur (S8) showed no antimicrobial activity in the FDA assay. However, sulfur did not dissolve in the acetone and cholesterol was observed to precipitate from the broth at a concentration of 10 mg/ml.

Chapter 6: Volatile organic compounds in the egg masses of molluscs 296

Table 6.8: The antimicrobial activity of some compounds found in the egg masses of marine molluscs. The compounds are all of synthetic origin and have been tested in the Fluorescein diacetate hydrolysis assay at a concentration of

10mg/ml. ++ indicates the complete suppression of FDA hydrolysis; + indicates reduced FDA hydrolysis compared to the control cultures; - indicates no reduction in FDA hydrolysis.

Compound Antimicrobial Activity E. coli S. aureus P. aeruginosa Cholesterol - - - Steric acid + + + Heptadecanoic acid + + - Oleic acid ++ ++ ++ Triolein + + - Sulfur - - - Chapter 6: Volatile organic compounds in the egg masses of molluscs 297

6.5 Discussion

6.5.1 Bioactive compounds from muricid egg masses The egg capsules of all muricids were found to contain tyrindoleninone (3;

Table 6.2), which is the major antimicrobial metabolite isolated from the egg mass of Dicathais orbita (refer to Chapter 5). The highly bacteriostatic compound tyriverdin (4) and the mildly antimicrobial oxygenation product 6- bromoisatin (7), were also isolated from the egg masses of several muricids

(Table 6.2). Consequently, these three compounds are likely to be responsible for much of the observed antimicrobial activity in the egg masses of muricid molluscs.

A number of additional brominated indoles were also observed in the muricid egg masses (Table 6.3). 6-Bromotryptamine derivatives have been previously isolated from species in a range of marine phyla (Fahy et al., 1991).

Antimicrobial activity has not been reported for any of the identified bromoindoles (14, 41, 42). However, a large number of halogenated indoles isolated from algae have been shown to possess antibacterial and antifungal activities (Alvarez and Salas, 1991). Several non-halogenated marine indoles also appear to have significant biological activities (Capon et al., 1986; Herb et al., 1990; Gil-Turnes et al, 1989; Kon-Ya et al., 1994; Davis and Bremner, in press). Two Mediterranean muricids were found to contain several nonhalogenated indoles (20, 48, 49, 51) in their egg masses. These include isatin

(48), a compound that has been implicated in the protection of crustacean eggs from fungal attack (Gil-Turnes et al., 1989) . This compound is also known to cause convulsions and narcosis in mice (von Muller, 1962). The Chapter 6: Volatile organic compounds in the egg masses of molluscs 298 remaining indoles identified in the muricid egg masses have not previously been isolated from a natural source and their pharmacological properties are not known.

The distribution of indigoid dye precursors in the Mediterranean muricid egg masses is unexpected, based on previous reports of the chromogens found in these species. Indigotin (7) has only previously been reported from

Trunculariopsis (Murex) trunculus (Elsner and Spanier, 1985; Michel et al.,

1992). Therefore the non brominated indole precursor 2-methylthio-3H-indol-

3-one (21, Scheme 6.1) was only expected to occur in the egg mass of this species. However, this compound was found in the egg mass of Ceratostoma

(Murex) erineceum, as well as T. trunculus. Similarly, isatin (48) could be expected to occur as an oxygenation product from the non brominated precursors 21and 23. However, this compound was only found in the egg mass of C. erineceum, a species which is thought to contain only the brominated chromogen 17 (Scheme 6.2; Prota, 1980) or possibly a second brominated chromogen 18 (Scheme 6.2; Baker, 1974). It now seems likely that C. erineceum also contains the chromogen 16 (Scheme 6.2) that has been reported from T. trunculus (Fouquet and Bielig, 1971).

The presence of 6-bromo-2-methylsulfonyl-3H-indol-3-one (22) was expected but not found in the egg masses of the two Mediterranean muricids

(Ceratostoma erineceum and Trunculariopsis trunculus). This compound is an intermediate precursor to Tyrian purple formed from the ultimate precursor 17

(Scheme 6.2), which is known to occur in these two muricids (Fouquet and Chapter 6: Volatile organic compounds in the egg masses of molluscs 299

Bielig, 1971; Baker, 1974; Prota, 1980). The 6-bromo-2-methylsulfonyl-3H- indol-3-one was not positively identified in the egg masses but it remains possible that it is one of the brominated indoles that did not produce a clear molecular ion in the mass spectrometer (refer to Table 6.2). This compound may also be fairly unstable resulting in the loss of a methyl sulfone. A significant quantity of methyl methane-thiosulfonate (53) was detected in the eggs of Trunculariopsis trunculus, which could result from a reaction between methyl sulphone and methane thiol from the tyriverdin or tyrindoleninone. The sulfoxides and dimethyl trisulphide (54) detected in the muricid egg masses are also likely to be decomposition products from the indigotin dye precursors.

The presence of 6-bromoindoxyl (14) was unexpected in the egg mass of D. orbita. This compound is an intermediate precursor of Tyrian Purple formed from the precursor 10 (Scheme 6.1), which has only been found in the hypobranchial glands of Trunculariopsis trunculus (Fouquet and Bielig, 1971;

Baker and Sutherland, 1968; Baker, 1974). The 6-bromoindoxyl (14), along with 6-bromo-2-methylsulfinyl-indol-3-one (41), 6-bromo-2-methoxy-indol-3- one (42) and 4-bromo anthranilic acid (43) are likely to be oxidation and decomposition products from tyrindoleninone (3) in the egg mass of D. orbita.

Freeze-drying appears to increase the oxidation of tyrindoleninone (3) to 6- bromo-2-methylsulfinyl-indol-3-one (41; Table 6.3), suggesting that this compound is also likely to be an artefact in the egg extract from Ceratostoma erineceum.

Chapter 6: Volatile organic compounds in the egg masses of molluscs 300

Freeze-drying the eggs of D. orbita also leads to the formation of an insoluble red substance. A similar substance was observed in the extracts from the freeze-dried eggs of the two Mediterranean species. This substance could be a mixture of indirubins (26-29), which are red compounds formed from the condensation of (bromo) indoxyl and (bromo) isatin (Clark and Cooksey,

1997). The removal of water from the egg capsules of muricid molluscs by freeze drying appears to increase the oxidative reactions to the indoxyl precursors, leading to a greater suite of products. Many of these products may not naturally occur in the muricid egg masses. Nevertheless, these artefacts do provide a greater range of potentially interesting drug leads and their relative stability and antimicrobial activity should be compared.

Some previous reports have suggested that molluscs in the genus Mitra also produce Tyrian Purple (Fox 1966; Ghiretti, 1994), although this has not been supported by any experimental data. The egg mass of the Australian species

Mitra carbonaria, was not found to contain any precursors to Tyrian Purple in this study (Appendix 6.1). However, the intracapsular fluid in the egg mass of this species does undergo colour change over time from white, through orange to a deep magenta (personal observation). This colour change proceeds extremely rapidly in aerobic conditions and leads to the production of an highly insoluble dye. Therefore, it is possible that M. carbonaria produces some related dye precursors to protect their egg masses. Further work on the bioactive compounds from the egg masses of Mitra carbonaria would have to be carried out under anaerobic conditions. Nevertheless, it Chapter 6: Volatile organic compounds in the egg masses of molluscs 301 appears likely that the precursors of Tyrian Purple are unique to the muricid molluscs and may be a characteristic feature of their egg masses.

Other brominated compounds from the egg masses of muricid molluscs include the di- and tribromo pyrazoles (44, 45) and/or imidazoles (46, 47;

Table 6.2). It will be necessary to synthesise these compounds to confirm their identity. The synthesis of di- and tribromoimidazoles has been widely patented (Sharnin et al., 1976; Bessonov and Tertov, 1977; Tomioka et al.,

1988; Toshihiko et al., 1998a,b), although these compounds do not appear to have been previously isolated from a natural source. Overall, simple imidazole and pyrazole derivatives appear to occur infrequently as secondary metabolites. However, novel pyrazole acids have also been isolated from a marine sponge (Parameswaran et al., 1997) and an imidazole derivative has been found in the nudibranch Phestilla melanobranchia (Okuda et al., 1982).

An imidazole with cytotoxic and antibiotic properties has been isolated from an ascidian (Copp et al., 1989). Interestingly, 3-methyl-5-phenyl-1H-pryazole

(52) has been found in krill (Gajowjecki, 1992) and a similar compound appears to occur in the egg masses of Ceratostoma erineceum (Appendix

6h).

The biocidal activity of the bromoimidazoles has been well established. 4,5-

Dibromoimidazole has been incorporated into a US patented herbicide that is effective in preventing the germination of weed seeds (Draber et al., 1998).

Tribromoimidazole was shown to be toxic when administered to rats, causing neuronal necrosis and chromatolysis in the brain (Verschoyle et al., 1984). Chapter 6: Volatile organic compounds in the egg masses of molluscs 302

Tribromoimidazole is used as the active ingredient in insecticides and aracicides (Tomioka et al., 1988; Toshihiko et al., 1998a,b). It is also used for the synthesis of a range of derivatives used in insecticides, fungicides and herbicides (Leone-Bay et al., 1986; Boots Pure Drug Co. Ltd., 1998; Rutz and

Gubler, 1998a;b; Susumu et al., 1998; Tomioka et al., 1998a;b; Veverka,

1998). Consequently, it is likely that the bromoimidazoles/pyrazoles found in extracts from muricid egg masses contribute to the antibiotic activity of these extracts (refer to Chapter 4).

6.5.2 Bioactive metabolites in Apysiidae egg ribbons The halogenated compounds (55-64, Table 6.4) found in the extracts from

Aplysiidae egg ribbons could be partly responsible for the observed antimicrobial activity in these egg (refer to Chapter 4). Small halogenated compounds isolated from red seaweed are known to be biocidal (Fenical,

1982). Furthermore, halogenated propanes and propenes (e.g. 59) are known to have mutagenic and cytotoxic effects (Watson et al., 1987; Lag et al.,

1994). All of the chlorinated compounds that have been identified do no appear to have been previously reported from a natural source. However, these compounds were not found in the solvent controls or the egg extracts from any of the other species that were analysed and thus appear to be genuine new natural products. The biosynthesis of similar volatile brominated metabolites has been reported from marine algae (Theiler et al., 1978;

Beissner et al., 1981).

Chapter 6: Volatile organic compounds in the egg masses of molluscs 303

It is possible that the chlorinated compounds found in the Aplysiidae egg ribbons are cleavage products of the halogenated terpenes or enynes that are commonly found in sea hares (refer to Faulkner, 1984; 1992 and references therein). There is no evidence of chlorinating enzymes occurring in

Opisthobranch molluscs and therefore these compounds are most likely to be dietary derived. Nevertheless, the presence of halogenated compounds in the egg ribbons of sea hares is surprising because previous reports have suggested that the egg masses do not contain the dietary derived metabolites found in the adults (Faulkner, 1992; Pennings and Paul, 1993; Pennings,

1994; Rocky de Nys, pers. comm.).

The gelatinous egg ribbons of species in the Aplysiidae family were also found to be dominated by a range of long chained saturated and unsaturated fatty acids (Figure 6.1, Tables 6.5, 6.6). These acids could be responsible for much of the antimicrobial activity observed in the lipophilic extract from these egg ribbons (refer to Chapter 4). In this study oleic acid was found to have significant antimicrobial activity (Table 6.8). Minimal antimicrobial activity was also observed with stearic and heptadecanoic acids, as well as the triglyceride triolein (Table 6.8).

Fatty acids and methyl esters could also be involved in the feeding deterrent activity of the egg masses from the Aplysiidae (refer to Chapter 3; Pennings and Paul, 1993; Pennings, 1994). Several fatty acids have been identified in the defensive droplets on the egg stalks of green lacewings (Eisner et al.,

1996), as well as in the nests of wasps (Dani et al., 1996). These defensive Chapter 6: Volatile organic compounds in the egg masses of molluscs 304 secretions were both found to act as deterrants to ants. Oleic and linoleic

(C18 dieneoic) acids isolated from the lacewing egg stalk fluid are also irritants to cockroaches (Eisner et al., 1996). The methyl ester of palmitic acid is found in the nest secretions of some wasps and also functions to deter ants

(Jeanne, 1970; Post et al., 1981; Henderson and Jeanne, 1989).

Several unidentified long chained unsaturated fatty acids were found in the egg masses of Aplysia juliana and Stylocheilus longicauda (Table 6.5). These could be C21 acids sequestered from marine algae. Fatty acids with a chain length of 21 are extremely uncommon in nature, having only been reported from marine microalgae (Mansour, 1998; Dr Peter Mansour, pers. comm.).

Alternatively, these acids could be unusual C20 or C22 acids, such as the unique cyclopropane containing C20 acid isolated from the sea hare

Bursatella leachii (Fenical et al., 1979).

Some of the observed fatty acids in the egg ribbons of the Aplysiidae could be breakdown products from larger lipids. In particular, a higher proportion of unsaturated fatty acids was found in the derivatised samples of the egg extracts (Table 6.5), suggesting these could be products of hydrolysis. The fatty acids components from glycosphingolipids, cholesteryl esters (24) and one diacylglyceryl ether (23) have been previous analysed in the egg ribbons of Aplysia spp. (Yamaguchi et al., 1992a;b; Miyamoto et al., 1998). However, these authors report primarily saturated fatty acid in the lipid metabolites, whereas a range of polyunsaturated acids from C16-C22 were found in this study. This could represent interspecies differences in the lipid content. Chapter 6: Volatile organic compounds in the egg masses of molluscs 305

Overall, it appears likely that the egg ribbons of the Aplysiidae are protected by a diversity of different bioactive metabolites (see also Yamazaki et al.,

1985; Kisugi et al., 1987; Miyamoto et al., 1988; Yamaguchi et al., 1992c).

Previous studies on the antimicrobial and cytotoxic glycoproteins from

Aplysiidae egg ribbons indicates there is significant interspecies variation in these compounds (Yamazaki, 1993). Greater variation in glycoprotein composition appears to occur between the different genera within the

Aplysiidae (Yamazaki, 1993). Likewise in this study, the fatty acid composition of the egg ribbons from Stylocheilus longicauda showed greater interspecies differences than the composition of fatty acids in the three Aplysia spp. (Table

6.5; 6.6). This is indicative of a relationship between biological diversity and the diversity of bioactive compounds within the Aplysiidae family.

6.5.3 Bioactive metabolites from other molluscan egg masses The egg capsules of Dicathais orbita were found to be exceptional in containing a high proportion of a single unusual metabolite (refer to Chapter 5;

Appendix 6.1). Most other molluscs appear to contain a large number of relatively minor metabolites in their egg masses. Consequently, the isolation and identification of the active components in the egg masses from other molluscs could be more complicated. The observed antimicrobial activity in these species could be due to an highly potent minor metabolite, the synergistic action of several metabolites or non-volatile organic components in the egg masses.

Chapter 6: Volatile organic compounds in the egg masses of molluscs 306

Fatty acids and/or the methyl esters of acids could be partially responsible for the antimicrobial activity in the egg masses of many molluscs. Extracts from the egg masses of all species examined were found to contain some fatty acids, with the exception of Mitra carbonaria, which was the only lipophilic extract found to have no antimicrobial properties (Chapter 4). However, fatty acids are less likely to play a significant role in the antimicrobial defence of leathery egg capsules than the gelatinous egg masses of marine molluscs.

The leathery egg capsules of the Neogastropods and the Littorinimorpha

Cabestana spengleri were all found to have a small proportion of exclusively saturated fatty acids (Figure 6.1). However, the unsaturated fatty acid, oleic acid was found to have significantly greater antimicrobial activity than the saturated fatty acids that were tested in this study (Table 6.8). Oleic acid was found in the egg masses of nearly all the gelatinous egg masses, as well as the egg capsules of the Cephalopod Sepioteuthis australis (Table 6.6).

The egg mass of the Opisthobranch Spurilla neopolitana was unusual in containing C15 fatty acids and several unidentified unsaturated acids. Fatty acids with an uneven chain length are generally uncommon in animals

(Tagliamonte and Tomassi, 1976). However, marine invertebrates can be the source of unusual fatty acids, some of which have demonstrated antibiotic properties (e.g. Carballiera et al., 1997). A large number of other oxygenated metabolites, such as alcohols, ketones and aldehydes were also found in the egg masses of several molluscs. These types of compounds could result from the oxidation of unsaturated fatty acids. Notably, aldehydes have been isolated from the defensive secretions of several insects (Eisner et al., 1996). Chapter 6: Volatile organic compounds in the egg masses of molluscs 307

Isopropyl myristate was also found in the defensive secretion of the egg stalk fluid from the green lacewing (Eisner et al., 1996). This compound was found in a seawater extract, as well as the egg masses of many molluscs (Appendix

6.8; 6.1). However, the seawater extract containing isopropyl myristate was not found to have antimicrobial activity in this study (Chapter 4).

Sterols were found in significant proportions in the egg masses of all molluscs that were examined (Figure 6.2). However, sterols are unlikely to be responsible for the antimicrobial activity in these egg masses because cholesterol did not effectively inhibit the growth of three test bacteria in the

FDA assay (Table 6.8). Cholesterol was the main sterol found in the molluscan egg masses, which is consistent with previous studies on molluscan sterols (refer to Figure 6.2; Bergmann, 1949; Idler and Wiseman,

1972). Nevertheless, a range of minor sterols were also found in the egg masses and previous studies on marine organisms have resulted in the isolation of novel sterols with significant cytotoxicity (Schroeder et al., 1980;

Guyot et al., 1983; Heltzel et al., 1994; Sheu et al., 1995; 1997). Antifeedant activity has also been reported for one sterol isolated from a nudibranch (Ayer and Andersen, 1982) and defensive roles have been proposed for the oxygenated sterols isolated from the egg ribbons of Aplysia juliana

(Yamaguchi et al., 1992). There is also some evidence to indicate that cholesterol has antifouling activity (Davis et al., 1991).

Sulfur and hexathiepane were found in the egg ribbons of the estuarine pulmonate Salinator fragilis (Table 6.7). The origin of these compounds Chapter 6: Volatile organic compounds in the egg masses of molluscs 308 appears to be the mud, which is incorporated into the egg ribbons of S. fragilis

(refer to Chapter 3). However, sulfur (S8) did not show any antimicrobial activity in the FDA assay (Table 6.7). Nevertheless, it remains possible that sulfur has antimicrobial activity in a more soluble form, such as S6, which was also found in these extracts (Table 6.7). Hexathiepane is likely to be a biologically active compound. The similar polysulfide lenthioine has been isolated as the antibacterial component of a mushroom (Morita and

Kobayashi, 1967) and a red seaweed (Wratten and Faulkner, 1976).

A large number of solvent contaminants were found in the egg extracts and it was sometimes difficult to determine which components were artefacts.

Future studies should use distilled solvents and solvent controls should be run from every batch of solvent used. The concentration of extracts injected into the gc/ms should also be standardised for the purpose of cross comparison.

Nevertheless, it appears that chloroform is a more suitable solvent than diethyl ether for use in gc/ms analyses. The diethyl ether was found to contain significant concentrations of several phenolic contaminants including the antioxidant butylhydroxytoluene (BHT). BHT is a known tumor promoter at high concentrations (Kahl and Kappus, 1993; Iverson, 1995) and has also been shown to enhance the antimicrobial activity of some marine terpene alcohols (Kubo et al., 1992). A dried down ether extract was not found to have antimicrobial activity in this study (Chapter 4). However, the synergistic effects of BHT could be responsible for some of the intraspecies variation in the antimicrobial activity of extracts from the egg masses of Conuber sordidus

(refer to Chapter 4), as well as the greater activity in a diethyl ether extract Chapter 6: Volatile organic compounds in the egg masses of molluscs 309 from Dicathais orbita egg masses, compared to the chloroform extracts (refer to Chapter 5).

6.6 Conclusion

The egg masses of marine molluscs contain a wide range of volatile organic compounds. Precursors of the ancient dye Tyrian Purple were found in the egg masses of six species of Muricidae. In most muricids, these precursors were accompanied by a range of additional brominated indoles, some of which could not be identified. Two Mediterranean muricids were also found to contain precursors of indigotin and several other nonbrominated indoles.

Brominated pyrazoles and/or imidazoles were also found in the egg masses of several muricids. The brominated compounds and other indoles are all likely to contribute the antimicrobial activity of muricid egg masses.

Different classes of compounds must be responsible for the antimicrobial activity in the egg masses from molluscs in other families. Halogenated compounds were only found in the egg masses of one other family of molluscs, the Aplysiidae. These were small, chlorinated compounds, that are most likely derived from a dietary source. Fatty acids could contribute to the biological activity in the egg masses of many species. In particular, a high proportion of unsaturated fatty acids was found in the extracts from some gelatinous molluscan egg masses and oleic acid was shown to have antimicrobial activity. Other significant volatile metabolites found in molluscan egg masses include cholesterol and sulfur. However, neither of these compounds appear to be responsible for the observed antimicrobial activity. Chapter 6: Volatile organic compounds in the egg masses of molluscs 310

Overall, it is likely that a greater range of biologically active compounds could be discovered from the study of a larger diversity of molluscan egg masses.

NOTE

This online version of the thesis may have different page formatting and pagination from the paper copy held in the University of Wollongong Library.

UNIVERSITY OF WOLLONGONG

Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 311

Chapter 7 General Discussion:

Bioactive Molluscan Resources and Conservation

7.1 Biodiversity and Bioresources

“ Mere Conchs! not fit for warp or woof!

Till cunning come to pound and squeeze

And clarify, - refine to proof

The liquor filtered by degrees,

While the world stands aloof.

And there’s the extract flasked and fine,

And priced and saleable at last!”

Robert Browning, 1855

As demonstrated in the above passage by Robert Browning, the value of many species on Earth is unlikely to be appreciated until they have a demonstrated resource value. Muricids were considered as merely snails not fit for consumption, until the extraction of the highly valued ancient dye, Tyrian

Purple. Murex molluscs were subsequently worshiped and became protected by laws under the Roman Empire (Baker, 1974; Hoffman, 1990). However,

Tyrian Purple is now only of historical interest and interest in the preservation Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 312 of the murex also appears to have faded. Along with many other marine molluscs, Muricids are currently under threat from the destruction of habitat, pollution, indiscriminate overfishing and lack of knowledge (Wye, 1996,

Ponder, 1998; Underwood, 1993).

Recently, the provision of natural resources from invertebrates and other biota, has been used as an argument for conservation (Roberts 1992; Beattie

1996). However, there has been some concern within the conservation movement that only a small proportion of species will turn out to be useful

(Ehrenfeld, 1988; Lawton, 1991). Many authors suggest that moral and ethical reasons for conservation are the most important. As stated by Ehrenfeld

(1988) “ the public must come to understand the inherent wrongness of the destruction of biological diversity”. While this may be true, Randall (1991) suggests that ethical arguments are unlikely to gain acceptance in a world that is dominated by the market place. Ultimately however, the commodity value of biodiversity will be much greater than is generally realised. Molluscs, for example, have adapted to a wide variety of life situations and the loss of any diversity will mean the loss of a library of adaptive information and potential resources (Benkendorff, in press). As pointed out by Norton (1989)

“every species illustrates an alternative means of sustaining life”.

In recent times there has been much focus on discovering new sources of antibiotics (Beattie, 1994; Cragg et al., 1997a). This study has demonstrated that egg masses of marine molluscs provide a novel source of potential antimicrobial agents (Chapter 4). In particular, a useful drug lead (tyriverdin) Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 313 has been isolated from egg mass of the southern Australian Muricid, Dicathais orbita (Chapter 5). There is a remarkable diversity of volatile organic compounds within molluscan egg masses (Chapter 6) and this chemical diversity is likely to increase with greater species diversity. Species from just one family, the Muricidae, were found to elaborate a variety of bioactive compounds (Chapter 6), which could provide a range of structural leads for drug development. However, different classes of chemical structures could be obtained by studying species with greater genetic dissimilarity, such species from different families (e.g. the Aplysiidae, refer to Chapter 6; Kisugi et al.,

1987; Miyamoto et al., 1988; Yamazaki, 1993). In order to obtain the optimal product, the study a range of different species is fully justified. The preservation of molluscan biodiversity can therefore be argued on utilitarian grounds alone.

One problem with using commodity value as an argument for conservation is that the economic criteria of value are fluid, and opportunistic in their practical applications (Ehrenfeld, 1988). However, this study demonstrates that new resources can be found in well-studied species and also, that new uses can be found for redundant natural resources. This may depend on being able to return to the source in order to study the natural processes that lead to the evolution of the resource. Previous studies have shown that the precursors of

Tyrian Purple are produced by the common dogwhelk Dicathais orbita (Baker and Sutherland, 1968; Baker and Duke, 1973a,b; Baker, 1974). However, the natural function of these compounds has remained unknown. A rationale approach to drug discovery and bioassay-guided fractionation lead to the Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 314 isolation of the potent antibiotic, tyriverdin, from D. orbita, in this study. It now seems likely that this immediate precursor to Tyrian Purple has evolved to protect the developing embryos of some Muricids against microbial infection.

The precursors of Tyrian Purple could, once again become a valuable commodity but this time as a novel lead for drug synthesis, rather than a luxury colorant. Clearly, human needs and values do change, but this should provide further incentive for the preservation of biological diversity. It has been suggested that all species could be assigned an ‘option’ value to allow for the

“possibility that a future discovery will make useful a species that we currently think is useless” (Norton, 1988).

7.2 The biorationale approach to drug discovery

“ The secret of finding something is knowing where to look”

Annon (quoted in Beattie, 1994).

It has been suggested that the chance of discovering new biological resources can be greatly improved by applying the knowledge of an organisms natural history and evolutionary biology (Beattie, 1994, 1996). This study provides support for the benefits of using a biorational approach towards the discovery of novel antimicrobial agents. Molluscan egg masses appear to be vulnerable to microbial infection and indeed the egg masses from a wide range of species were found to contain antimicrobial properties

(Chapter 4). In fact, antimicrobial properties appear to be widespread in the egg masses of marine invertebrates (Chapter 4; Kamiya et al., 1984, 1988;

Yamazaki et al., 1984, 1985; Matsunaga et al., 1986; Kisugi et al., 1987; Gil-

Turnes et al., 1989; Gil-Turnes and Fenical, 1992; Yamazaki, 1993). This Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 315 suggests that microbial infection at the egg stage is a significant selective pressure leading to the evolution of chemical defence.

The egg mass of Dicathais orbita and the honeycomb of bees provide an interesting example of convergent evolution. Both Aristotle (Historia

Animalium Vol. II, V. XV) and Pliny the Elder (Historia Naturalis Cap. XXXVIII.

Sect. 62. 133.) describe the “honeycomb-like exudate” of muricids. However, these early natural historians did not correctly identify the exudate as reproductive material. Nevertheless, this analogy is highly appropriate. Like honeycomb, muricid egg masses are the structurally complex brood cells of invertebrates and both appear to be sterilised with natural antibiotics (Chapter

5, Blum et al., 1959). The evolution of a similar reproductive strategy in species from two phyla, that occupy completely different habitats, is quite remarkable and indicates that the success of invertebrate brood cells could depend on the evolution of an antimicrobial defence. Other examples of convergent evolution could also provide useful leads for the discovery of novel biological resources. As stated by Norton (1989) “Every wild species is a repository of analogies that inform our ongoing struggle to survive”.

Other selective pressures that could led to the evolution of bioactive metabolites in the marine environment include predation and surface fouling

(refer to Bakus et al., 1986, Paul, 1990, Pawlik, 1993). However, physical, behavioural and physiological adaptations could also be used to reduce the impacts of these factors on molluscan egg masses (Chapter 3). Physical defence, such as the leathery egg capsules of Neogastropods and the shells Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 316 of many molluscs, may negate the requirement for chemical feeding deterrents (Chapter 3; Faulkner, 1992a). Nevertheless, the absence of activity in one biological assay should not be used as a general indicator to the absence of biologically active compounds (see also Thompson et al., 1985).

For example, some species, such as Dicathais orbita, appear to use physical deterrents as the primary mechanism to reduce predation on their egg masses (Chapter 3), while still producing antimicrobial metabolites (Chapters

4&5).

7.3 An interdisciplinary approach

There are strong benefits in taking an interdisciplinary approach to drug discovery. Information from the biological sciences can be used target those organisms most likely to yield an appropriate natural product and to select species that are able to withstand collection. However, the microscale analytical techniques available in chemistry are essential for the isolation and identification of the active components. Elucidation of the chemical structure of bioactive compounds may then permit synthesis for secondary pharmacological testing. The relatively uncomplicated structure of Tyriverdin is advantageous because it facilitates large-scale production without the need for further harvesting from the environment. Knowledge of the structure of

Tyriverdin from the literature has also reduced the amount of sample that was required for structural elucidation in this study. Computer modelling can then be used to generate chemical analogues of superior clinical efficacy and to examine structure-function relationships (refer to Torkamani et al., 1998).

However, commercial applications for more complicated natural products may Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 317 require further input from the biological sciences, to explore alternative methods of large-scale production, such as biosynthesis using combinatorial biology or mariculture (Colwell, 1983; Flam, 1994.).

The methods used to screen for biological activity are also an important consideration for bioprospecting. The methods will influence the amount of sample that is needed and therefore should be optimised to minimise the sample requirements. In recent years, the quality and efficiency of bioassays has greatly improved. It has been suggested that 500g is generally sufficient plant material for pharmaceutical evaluation (Asebey and Kempenaar, 1995).

The contract guidelines specified in the Manila Declaration (1992, Appendix 2) suggest that “the amount of material collected for initial screening should not normally exceed 100-500 grams”. Given the efficiency of modern screening technologies 500 grams is likely to be more than sufficient for the initial screening process.

In this study, I chose to use two complementary techniques to screen for antimicrobial activity (Chapter 4). Using the zone of inhibition assay I was able to test small samples of egg material (~1cm pieces) without the need for any sample preparation. Whereas, the FDA assay can be run in a 96 well microtitre plate and requires only 40mg of extract to test three replicates against four different pathogens, at a maximum concentration of 10mg/ml. In general, I found that 40mg of extract could be obtained from less than 20g of egg material. The minimal sample requirements for these assays made it possible to screen the egg material from a range of different molluscs, Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 318 including some species that were found to be uncommon. Rare species could also be tested when they deposited egg masses in aquaria after collection for taxonomic purposes.

Analytical techniques such as gas chromatography/mass spectrometry

(GC/MS) can also be useful rapid screening methods, to prevent the repeated isolated of the same compounds in different species. This technique requires only micro gram quantities of extract for analysis of the volatile organic constituents. Using this method, I was able to screen the egg masses of 23 species of mollusc (Chapter 6). The active constituents of Dicathais orbita egg masses were found in the egg masses of all the muricids that were examined.

However, a range of other potentially bioactive compounds were also tentatively identified (Chapter 6).

Whilst the initial screening for biological activity may only require small amounts of sample, the isolation and structural elucidation of active components will typically require a great deal more. It is in the recollection of organisms for detailed chemical and pharmacological examination that there is a real risk of overcollection. The actual amount of sample required will vary depending on the amount of the active compound present in the organism and the complexity of its chemical structure. Some organisms contain only tiny amounts of very potent chemicals, which essentially require huge collections. For example, Garson (1996) reports that in one study 2400 kg of sponge was collected to isolate only 1mg of an anticancer chemical. Further collections should therefore be justified, not only by the demonstrated Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 319 biological activity of the species but also by the ability of the species to recover after a large collection. It has been suggested that an environmental impact study should be carried out for the large-scale harvesting of marine resources (Garson, 1995; Workshop in Kuala Lumpur, 1996). If the species is locally abundant with a wide distribution it should be possible to collect sufficient material from only a small proportion of the population with minimal environmental impact. Alternatively, a naturally recruiting aquaria population could be used.

Preliminary screening in the egg masses of marine molluscs indicated that the

Muricids would be a good target for future research. The egg masses from these species had reasonable antimicrobial activity (Chapter 4) and GC/MS revealed the presence of high proportions of unusual brominated indoles

(Chapter 6). However, only two species of Muricidae were found to be locally abundant with wide distributions in southeast Australia (Chapter 2). Of the two, Dicathais orbita was selected for further research, because of its’ large size and the comparative ease of collecting the egg material. In total 300g of

Dicathais orbita egg mass was collected, which represented only a tiny fraction of the total population. From this 300g, I was able to isolate, structurally elucidate and characterise the antimicrobial activity of three small organic molecules. This demonstrates that pharmacologically useful compounds can be discovered with little impact on the source organism.

Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 320

7.4 Sustainable bioprospecting

Chemical prospecting for pharmaceuticals in natural organisms has been both championed as a tool for conservation and criticised for not being environmentally sustainable. However, there has been little attempt to address the ways in which bioprospecting can contribute to conservation or the means by which bioprospecting can be sustainable. Clearly, bioprospecting will have no long-term potential unless there is an intensified worldwide effort to conserve biodiversity (Eisner and Meinwald, 1990).

Consequently, bioprospecting does offer an incentive for conservation. But bioprospecting can only be considered compatible with the objectives of conservation if it can be shown to have no negative impact on the environment.

In order to ensure that bioprospecting is sustainable we may need to make some changes in the way that biological data is used and reported in natural products research. The ecological impact of biological collections depends primarily on the amount of specimen collected and the rarity of the species.

Rarity may be difficult to assess for many marine invertebrates (Chapman,

1998) and in these circumstances, knowledge on the local distribution and abundance of the species is important (refer to Chapter 2). Perhaps if researchers become aware of the value of this type of biological data it will be reported in the literature on a more regular basis.

The concerns surrounding sustainable bioprospecting could extend into all fields of science that require the collection of organisms from the natural Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 321 environment (refer to Chapter 1). However, these concerns have only been voiced recently with regards to natural products chemists. This is probably because it is assumed that biologists recognise the importance of natural history and ecological data. It may therefore be appropriate to develop a Code of Ethics for the collection of biological specimens for natural products chemists.

For example, a bioprospector should:

1) know or obtain the correct taxonomy of the species they are collecting.

2) have a basic understanding of the organisms natural history, particularly

its local abundance and distribution.

3) be aware of the potential impacts associated with collecting the specimen

and report any unforseen impacts, including destruction to habitat.

4) never take more sample than is required.

5) only take a small proportion of any one population.

6) lodge a voucher specimen in an appropriate institute accompanied by

information on the date and place of collection, as well as any other local

observations.

Similar codes of conduct are produced in guides for shell collectors and by

Malacological Societies to ensure minimal environmental damage is done by shell collectors (e.g. Wilson, 1993, pp 14; The Hawaiian Malacological

Society, reprinted in Wye, 1996, pp 21; The Malacological Society of

Australasia, 1998). There is some scepticism over the efficacy of self- administered rules such as a code of ethics. However, bioprospectors are Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 322 unlikely to deliberately cause environmental harm and it is not in their interests to do so. The main purpose of a code of ethics for collecting biological specimens would be to draw the attention of natural products chemists to important biological considerations.

7.5 Bioprospecting and conservation

There are two main things that are required for effective conservation; knowledge and political will (Wilson, 1993). The political will necessary for appropriate environmental management stems from a well-informed and environmentally conscious community (Wilson, 1993). Chemical prospecting can help raise community awareness about the need for conserving biodiversity and acts to raise the profile of otherwise undesirable or insignificant organisms. For example, molluscs (slugs and snails), are typically regarded as pests and carriers of disease (e.g. Stansic, 1998). However, this study has demonstrated that molluscs are a potentially valuable source of antibiotics. It is currently difficult to attract media attention to push the case for the importance of invertebrates in nature conservation (Smith, 1997).

However, my experience indicates that the media is very interested in stories involving the potential medicinal value of marine molluscs. Ultimately, this type of media coverage could act to replace feelings of disgust for organisms such as “slugs and snails”, with a sense of appreciation. Interestingly, species of medicinal value are also specifically listed as important for biodiversity and bioresource management in the International Convention on Biological

Diversity (1992, Annex 1) and the National Strategy for the Conservation of

Australia’s Biological Diversity (1998, Objective 1.1). Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 323

The development of species inventories and an understanding of the ecological processes that influence species diversity, are also essential for wise environmental protection (Norse, 1993; Wilson, 1993; Stork, 1994). In this study, the search for new antibiotics has triggered the development of species inventories along the Wollongong Coast, NSW, Australia (Chapter 2).

This information has increased our knowledge on the distribution and abundance of the local marine molluscs and should ultimately contribute to marine conservation in the region. Furthermore, this study has shown that rapid biodiversity assessment can be an effective means of identifying

‘hotspots’ of molluscan diversity in the intertidal zone (Chapter 2). These methods are unlikely to produce a comprehensive species list, although they can be used effectively to assess the relative biological significance of different sites along the coast. Breeding sites can also be rapidly located for those species that deposit benthic egg masses in the intertidal zone. An assessment of the geographical features at highly biodiverse sites could provide further information about the habitat requirements influencing molluscan diversity (Chapter 2).

7.6 Management recommendations

Conserving marine biological diversity not only requires political advocacy and expanding the knowledge base, but also includes various kinds of planning to regulate threats and establish protected areas (Norse, 1993). Marine reserves in particular, are considered crucial for biodiversity conservation and resource management (Fairweather and McNeill, 1991; Norse, 1993; Allison et al., Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 324

1998). Based on the findings from this study, the establishment of an intertidal protected area at Bass Point is highly recommended for effective marine conservation in the Wollongong Region. The intertidal reefs on the north side of Bass Point were found to be a ‘hotspot’ for molluscan diversity and include many species that are representative of the other reefs in the region (Chapter

2). Furthermore, Bass Point supports many rare species and is used as a breeding ground by a wide variety of molluscs (Chapter 2). These features are all regarded as important criteria for the selection of marine protected areas

(Fairweather and McNeill, 1991; Norse, 1993, Environment Australia, 1998).

Nevertheless, reserves are not sufficient for conservation in the marine environment, because they can not be isolated from all critical impacts

(Allison et al., 1998). Consequently, an integrated approach to marine management is required, including sensible development decisions all along the coast. For example, this study indicates that suitable breeding habitat for many intertidal molluscs on the highly wave exposed, southeast Australian coastline, could be more restricted than previously thought (refer to Ponder,

1998). Many molluscs appear to require submerged boulders in sheltered intertidal areas for the deposition of benthic egg masses (Chapter 2).

However, undisturbed sheltered intertidal reefs are uncommon in the

Wollongong region because these sites are also the preferred locations for marina developments (Marine Pollution Research, 1995; Benkendorff, in press). It is therefore recommended that the availability of suitable breeding habitat should be taken into consideration in future marina developments. In Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 325 particular, a cautious approach should be taken to the Shell Cove Marina in the Bass Point embayment.

It is well recognised that there is often insufficient scientific evidence available to make sensible development decisions. Consequently, it is essential that the precautionary principle is implemented in environmental decision making

(refer to Gullett, 1997). For example, it is uncertain whether the Shell Cove marina development will have a detrimental impact on marine biological diversity. However, given the apparent value of this unusually pristine marine environment, one could reasonably argue that the risk is too great to take.

Unfortunately however, the value of the natural marine environment was not recognised at the Shell Cove commission of inquiry (refer to Simpson and

Train, 1996; Benkendorff, in press) and there is no opportunity for a reassessment of the decision, within the NSW planning legislation

(Environmental Planning and Assessment Act, 1989). This suggests that it may be necessary to implement changes in the legislation to accommodate new scientific information and the precautionary principle. It also reinforces that scientists should act on their subjective feelings before it is too late. The destruction of an important educational resource (refer to Benkendorff and

Ayre, 1998) should be regarded as serious and as stated by Soulé (1986b) “In many situations conservation biology is a crisis discipline”.

7.7 Conclusion

Clearly, the discovery of biological resources can only be used as a tool for conservation if bioprospecting is conducted in an ecologically sustainable Chapter 7. General Discussion: Bioactive Molluscan Resources and Conservation 326 manner. However, I believe that bioprospecting can not only be sustainable but it can be used to help attain the objectives of conservation. Bioprospecting can contribute to the conservation of natural resources through; 1) financial benefits; 2) triggering biodiversity research; and 3) providing a potential value for a great range of organisms. The opportunity for obtaining a novel resource, with low environmental impact, may strengthen the case for maintaining natural ecosystems in the face of competing uses (refer to Farrier and Tucker, 1998). Only a small proportion of species may produce bioactive chemicals that can be developed into a commodity. However, human needs and values change, such that all species have ‘option’ value, irrespective of their intrinsic value. Consequently, it is inappropriate to rule out any species as not worth saving.

NOTE

This online version of the thesis may have different page formatting and pagination from the paper copy held in the University of Wollongong Library.

UNIVERSITY OF WOLLONGONG

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NOTE

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UNIVERSITY OF WOLLONGONG

Chapter 1: Appendix 1.1 387 Chapter 1: Appendix 1

Appendix 1.1

Table 1.1a: Summary of the data reported on specimens collected for a range of studies on natural products from marine molluscs. These studies represent a sample of the papers that have been published over the last 40 years. They were not specifically selected but rather incorporate all of the papers on marine molluscs that were read during the course of this study.

Journals Total No. Amount Collected reported Abundance reported Lab raised references Yes No Sometimes Yes No Sometimes Total 74 34 38 2 9 62 3 1/31 Chemistry 50 29 19 2 3 46 1 0 Biochemistry 16 3 13 0 2 14 0 0 Exp. Biology 9 2 7 0 5 2 2 1/31 J. Am. Chem. Soc. 10 5 5 0 10 0 Tetrahedron lett. 10 5 5 1 9 0 J. Org. Chem. 13 9 4 0 13 0

1 One species out of three used in the study, was collected from naturally recruiting populations in aquaria. Chapter 1: Appendix 1.1 388 Table 1.1b: Biological collections used in a sample of previous studies published on natural products from marine molluscs (arranged by year of publication). NR indicates not reported. Reference Species Amount Abundance Biological activity collected Whittaker, 1959 Thais floridana NR NR “biologically active” referenced Biochemistry Journal Whittaker, 1960 Thais lapillus NR NR Neuromuscular blocking action- referenced Ann. NY Academy of Science Murex fulvescens NR NR Urosalpinx cinereus NR NR Yamamura and Hirata, 1963 Aplysia kurodai 3kg dried NR - Tetrahedron Baker and Sutherland, 1968 Dicathais orbita NR NR - Tetrahedron Letters Thompson, 1969 Pleurobranchus peroni NR commonest intertidal “probably defensive” cites previous fish feeding Australian Journal of Zoology pleurobranchid in NSW trials Cypraea cladestina NR NR Cypraea spadicea NR NR Roseghini, 1971 Thais haemastoma NR NR Activity on frog rectus muscle- no methods Experimentia Baker and Duke, 1973a Dicathais orbita NR NR - Tetra. Letters Mancinella keineri NR NR Mancinella distinguenda NR NR Baker and Duke, 1973b Dicathais orbita 93.5 glands NR - Aust. J. Chem. (5.805g) Mancinella bufo 0.5-1g NR Mancinella keineri 0.5-1g NR Mancinella distinguenda 0.5-1g NR Stallard and Faulkner, 1974 Aplysia californica NR Common - Comp. Biochem. Physiol. B Kato and Scheuer, 1974 Stylocheilus longicauda NR NR Toxic to mice, referenced J. Am. Chem. Soc. Kato and Scheuer, 1975 Stylocheilus longicauda 5000 animals NR Toxic to mice Pure and Applied Chemistry (50kgs) Chapter 1: Appendix 1.1 389

Table 1.1 Cont. Reference Species Amount collected Abundance Biological activity and other notes Marthy, 1976, Nature Loligo vulgaris eggs NR Common Tranquillising effect– methods detailed. Vanderah and Schmitz, 1976 Aplysia dactylomela 3.5Kg dry NR - J. Org. Chem. (after extraction) Baker and Duke, 1976 Dicathais orbita 500 glands NR - Tetra. Lett. Mancinella keineri 4.96g dried glands NR Rose et al., 1978. Stylocheilus longicauda 8.5kg Temporally abundant Nontoxic; no methods J. Am. Chem. Soc. Hagadone et al., 1979 Phyllidia varicosa NR NR Toxic to fish and crustaceans - previous Helvetica Chimica Acta publication Hollenbeak et al., 1979 Aplysia dactylomela NR2 NR Cytotoxic – assays referenced Tetrahedron Schmitz and Hollenbeak, 1979 Aplysia dactylomela NR1 NR Marginal cytotoxicity- Referenced J. Org. Chem Gopichand and Schmitz, 1980 Bursatella leachii pleii 115 animals NR - J. Org. Chem Schulte et al., 1980 Hypselodoris godeffroyana 1 individual ‘large populations’ Fish antifeedant activity – details to be Helvetica Chimica acta Chromodoris maridadilus 3 individuals reported Kamiya and Shimizu, 1981 Aplysia kurodai egg mass NR NR, all eggs Agglutinating vertebrate erythrocytes – Bulletin of the Japanese Society of Aplysia juliana egg mass NR collected from tanks methods described Scientific Fisheries Aplysia dactylomela eggs NR Rinehart et al., 1981 199 species NR NR 16% antimicrobial activity Pure & Appl. Chem. 163 species 5% cytotoxic 20 species 16% antimicrobial activity 21 species None had antiviral activity 21 species 33% cytotoxic methods referenced Ayer and Anderson, 1982 Aldisa sanguinea cooperi NR NR Antifeedant - goldfish assay – Tetrahedron Letters methods referenced Hellou et al., 1982 Cadlina luteomarginata ~ 309 individuals3 NR Antifeedant - goldfish assay – Tetrahedron methods referenced

2 The dry weight of glands after extraction is reported as 459g in both of these studies, it is likely that different fractions of the same sample were used. 3 It is possible the same specimens were used for the extraction of the different compounds in these two studies but if so this is not made clear. Chapter 1: Appendix 1.1 390

Table 1.1 cont. Reference Species Amount collected Abundance Biological activity Thompson et al., 1982 Cadlina luteomarginata > 330 individuals2 NR Antifeedant activity - goldfish and wooly Tetrahedron sculpin, methods described in detail Biskupiak and Ireland, 1983 Siphonaria pectinata NR NR Antimicrobial activity – Tetrahedron Letters no methods reported. Cimino et al. 1983 Dendrodoris limbata 7 individuals NR Fish antifeeding activity – Science previous publication Hochlowski and Faulkner, 1983, Siphonaria diemensis NR NR Antimicrobial activity – disc diffusion assay; Tetrahedron Letters Siphonaria sp. (4) NR NR Inhibition of cell division – sea urchin assay

No antimicrobial activity. (methods not detailed or referenced) Hochlowski et al., 1983 Siphonaria denticulata 340 individuals NR Ichthyotoxic towards goldfish; not J. Am. Chem. Soc. antimicrobial – no methods reported. Capon and Faulkner, 1984 Siphonaria lessoni NR NR - J. Org. Chem. Hochlowski et al., 1984 Siphonaria zelandica >1000 individuals NR - J. Am. Chem. Soc. Siphonaria denticulata (combined) Siphonaria atra ~100 NR Siphonaria laciniosa NR NR Siphonaria normalis ~200 NR Kamiya et al., 1984 Aplysia kurodai egg mass NR NR Antibacterial – turbidometric assay Experientia Yamazaki et al., 1984 Aplysia kurodai egg mass NR NR Cytolytic activity; cytolytic and metabolic Gann (Japanese J. Can. Res.) assay detailed. Elsner and Spanier, 1985 Murex trunculus NR NR - Proc. 7th Intern. Wool and Text. Conf. Yamazaki et al., 1985 Aplysia kurodai egg mass NR NR Antineoplastic – cytolytic assay detailed FEBS

Chapter 1: Appendix 1.1 391

Table 1.1 cont. Reference Species Amount collected Abundance Biological activity

Carté et al., 1986 Chromodoris funerea 8 NR Sponge derived metabolites J. Org. Chem. Ichiba and Higa, 1986 Aplysia dactylomela 1 specimen, 290g NR Antibacterial J. Org. Chem. Ichthyotoxic against guppies; No methods Manker and Faulkner, 1986 Siphonaria maura 230 individuals NR Mild antimicrobial activity – no methods J. Org. Chem. Matsunaga et al., 1986 Dendrodoris nigra eggs NR NR No antifungal activity J. Am. Chem. Soc. Unidentified nudibranch egg mass 120g, 12 pieces4 NR Antifungal activity; disk diffusion assay Pawlik et al., 1986 Collisella limatula (limpet) >400 Abundant Antifeedant - ecologically relevant predators Mar. Ecol. Prog. Ser. Roesner and Scheuer, 1986 Hexabranchus sanuineus NR NR Antitumour activity - acknowledged J. Am. Chem. Soc. egg ribbons Antimicrobial activity - disk diffusion assay Young et al., 1986 Onchidella borealis NR Abundant at some Repellent towards intertidal predators – Biol. Bull. sites methods detailed Cimino et al., 1987 Bursatella leachii leachii 2 specimens NR - J. Org. Chem. Bursatella leachii savignyana 100 animals Kisugi et al., 1987 Aplysia kurodai egg mass NR NR Antitumor activity but not cytolytic, inhibition Cancer Research of DNA, RNA and protein synthesis; also active in vivo in mice. Methods detailed. Kusumi et al., 1987 Aplysia kurodai NR NR Cytotoxic; no methods J. Org. Chem. Pettit et al., 1987 Dolabella auricularia ~ 1000kg NR Antineoplastic - referenced J. Am. Chem. Soc. Alam et al., 1988 Planaxis sulcatus 7kg NR Cytotoxic - no methods. Steroids Cimino et al., 1988 Umbraculum mediterraneum 1 specimen Rare Ichthyotoxic - mosquito fish. Methods Tetrahedron letters referenced Kamiya et al., 1988 Aplysia juliana eggs and adults NR NR Antibacterial and antineoplastic - Nippon Suisan Gak. Turbidimetric assay. Methods detailed

4 The authors report that 12 pieces of these egg masses were extracted. It is not clear if these ‘pieces’ are 12 individual egg ribbons or 12 portions of fewer egg ribbons. Chapter 1: Appendix 1.1 392

Table 1.1 cont. Reference Species Amount collected Abundance Biological activity Source Manker et al., 1988 Siphonaria denticulata 46 individuals NR - J. Chem. Soc. Miyamoto et al., 1988a Aplysia kurodai NR NR Laxative lipid; referenced Leibigs Ann. Chem. Miyamoto et al., 1988b Aplysia juliana 617 (300kg) NR - Leibigs Ann. Chem. Bobzin and Faulkner, 1989 Chromodoris norrisi 3 specimens NR Antifeedant - Rainbow Wrasse, methods ref. J. Org. Chem. Antimicrobial -disk diffusion assay, no details. Manker and Faulkner, 1989a Siphonaria baconi 403 individuals NR - J. Org. Chem. Manker and Faulkner, 1989b Siphonaria maura 370 individuals NR - J. Org. Chem. Pettit et al., 1989 Dolabella auricularia NR NR Cytolytic – no methods J. Am. Chem. Soc. Bai et al., 1990 Dolabella auricularia NR5 NR Cytostatic activity – interference with microtubule Biochem. Pharm. assemblage. Methods detailed Kigoshi et al., 1990 Aplysia kurodai NR NR Cytotoxic - no methods Tetrahedron letters

Ojika et al., 1990 Aplysia kurodai 15.3kg wet NR Phospholipase A2 activity - no methods Tetrahedron letters Paul et al., 1990 Nembrotha spp. NR Common Feeding deterrents - reef fish Mar. Ecol. Prog. Ser. Yamazaki et al., 1990 Aplysia kurodai NR6 NR Antibacterial activity - absorbtion method. Bacteriostatic Developmental and activity - DNA and RNA synthesis assay detailed. Comparative Immunology Dolabella auricularia NR5 NR No antimicrobial activity Cytolytic activity referenced. Michel et al., 1992 Murex trunculus Several hundred Large groups - J. Soc. Dyers Colourists Murex brandaris As above + 30 from NR market

5 This is primarily a synthetic paper but some of the natural product was used for comparison. 6 These studies were conducted on the purple fluid exudate and probably did not involve killing the animals. Chapter 1: Appendix 1.1 393

Table 1.1 cont. Reference Species Amount collected Abundance Biological activity

Yamaguchi et al., 1992 Aplysia juliana egg masses 3.45kg wet NR phosphonoglycosphingolipids Biochem. Biophysica Acta Yamaguchi et al., 1992b Aplysia juliana egg masses 3.5kg wet NR Isolation of sterols Chemistry Letters Yamaguchi et al., 1992c Aplysia juliana egg masses 3.68g wet NR phosphonoglycosphingolipids Biochem. Biophysica Acta Pennings and Paul, 1993 Stylocheilus longicauda + eggs From aquaria Circumtropical Locations of sequestered metabolites Marine Biology Dolabella auricularia + eggs As above and field Common Aplysia californica + eggs NR NR Yamazaki, 1993 Aplysia kurodai NR NR Antitumor and Antimicrobial; referenced Comp. Biochem. Physiol. C Aplysia juliana NR NR Dollabella auriculata NR NR Pennings, 1994 Aplysia juliana + eggs NR Circumtropical Antifeeding activity – reef fish and crabs J. Exp. Mar. Biol. Ecol. Aplysia kurodai NR NR Aplysia oculifera NR NR Dolabella auricularia NR NR Miyamoto et al., 1995 Aplysia parvula 65 individuals NR Ichthyotoxic -mosquito fish no methods Tetrahedron Letters (320g) Rogers et al., 1995 Aplysia parvula NR Relatively uncommon, Some non destructive experiments but J. Exp. Mar. Biol. Ecol. Aplysia juliana NR Abundance mapped also extractions Yamada et al., 1995 Aplysia kurodai eggs 1kg NR Phosphonoglycosphingolipid J. Biochem. De Nys et al., 1996 Aplysia parvula Not clear temporally abundant Sequestered defence – predator Mar. Ecol. Prog. Ser. deterrent referenced Fernández et al. 1996 Onchidium sp. 3kgs (~10) NR Cytotoxic – bioassay directed J. Am. Chem. Soc. fractionation, no methods Clark and Cooksey, 1997 Nucella lapillus NR NR - J. Soc. Dyers and Colorists Spinella et al., 1997 Aplysia depilans 73 individuals NR Ichthyotoxic, Gambusia affinis – Journal of Organic Chemistry methods referenced

Chapter 1: Appendix 1.1 394

Appendix 1.2

Table 1.2a: Summary of the data reported on specimens collected for a range of biological studies on marine molluscs.

Journals Amount reported Abundance reported Distribution Lab raised Yes No Sometimes Yes No Sometimes Yes No All Biology 19 16 5 7 28 5 10 30 1 Natural history 11 4 5 4 12 4 8 12 0 Experimental biology 7 8 0 3 11 1 2 12 1 Biochemistry 1 5 0 0 6 0 0 6 0

Table 1.2b: Biological collections used in previous studies published on marine molluscs in a range of natural history, ecology, marine biology and biochemistry journals. NR refers to not reported.

Reference Species Amount collected Abundance Purpose of Collection/ Fate of organism

Anderson, 1959 Cymatilesta (Cabestana) 1 egg mass? Considerable Reproduction and life history Proc. Linnean Soc. NSW spenglerii numbers Anderson, 1965 Xenogalea labiata 1 egg mass NR Description of egg masses. Mostly adults Proc. Linnean Soc. NSW Bedeva hanleyi numerous capsules NR collected and spawned in aquaria. Some Morula marginalba NR Common egg masses collected from field. Nassarius particeps NR NR Siphonaria denticulata NR Common Flower et al., 1969 Buccinum undatum NR NR Study proteins in egg capsules J. Ultrastructure Research egg capsules Laxton, 1969 Cabestana spengleri NR NR Reproductive behaviour and anatomical Zool. J. Linn. Soc. Mayena australasia NR NR descriptions Monoplex australasiae NR NR Charonia sp. NR NR

Chapter 1: Appendix 1.1 395

Table 1.2b cont. Reference Species Amount collected Abundance Purpose of Collection/ Fate of organism

D’Asaro, 1970 Certhium auricoma NR Rare Description of egg masses and notes on Bull. Mar. Sci. C. literatum NR Very common spawning. Strombu gallus 2 NR Cypraea spurca acicularis NR NR Murex florifer 1 NR Murex pomum NR NR Thais rustica NR Moderately com. Cantharus tinctus NR NR Leucozonia nassa NR NR Fasciolaria tulipa NR Common Pleuroploca gigantea NR Relatively Vasum muricatum Adults NR common 1 egg mass NR Xancus angulatus 1 egg mass NR Oliva sayana NR NR Mitra nodulosa NR NR Prunum apicinum NR NR Conus spurius atlanticus NR NR Conus regius Several individuals + 1 Large population egg mass Price and Hunt, 1974 Buccinum undatum Eggs NR NR Fluorescent components in egg capsules Comp. Biochem. Physiol. Phillips, 1975 Cronia avellana NR NR Notes on egg capsules. Some C. avellana J. Malac. Soc. Aust. Cronia pseudamygdala NR NR egg capsules deposited in WA museum Spight, 1977 Thais lamellosa 3 groups out of 197 egg 50 breeding Spawning behaviour Evolution egg capsules masses groups av. 145 snails Mapstone, 1978 Siphonaria diemenensis NR NR Description of spawn + development J. Malac. Soc. Aust. S. baconi NR NR Adults and spawn of both Creese, 1980 Siphonaria denticulata Several + egg ribbons Common and Reproductive cycles Aust. J. Mar. Freshw. Res. Siphonaria virgulata 20 widespread Chapter 1: Appendix 1.1 396

Table 1.2b cont. Reference Species Amount collected Abundance Purpose of Collection/ Fate of organism

Perron, 1981 Conus spp. (10) 104 egg masses NR Reproductive energy The American Naturalist Brenchley, 1982 Ilyanassa obsoleta NR Abundant Predator experiments – lab and field Mar. Ecol. Prog. Ser. Perron, 1982 Conus pennaceus 140 + egg masses NR Reproductive effort (ashed) Marine Biology C. abbreviatus 8 + eggs NR C. flavidus 5 + eggs NR C. quercinus 17 NR Pechenik et al., 1984 Nucella lapillus NR NR Nutritional and antimicrobial properties of Marine Biology egg capsules Hoagland, 1986 Crepidula fornicata 40 adults + 25 broods NR Encapsulation and brooding; fate not American Malacological Bulletin C. fornicata + C. plana + hundreds of brooding reported C. convexa females NR D’asaro, 1986 Ticolia affinis ‘populations’ NR Laboratory spawning Amer. Malac. Bull. T. thalassicola T. bella Puperita pupa Smaragdia viridis Littorina mespillum Alvania auberiana Rissoina catesbyerea R. bryerea Zebina browniana Rissoella caribaea Caecum nitidum Marginella aureocincta Granulina ovuliformis Eyster, 1986 Coryphella salmonacea Eggs NR NR Ultrastructure of capsule walls Amer. Malac. Bull. Aeolidia papillosa Hermissenda crassicornis

Chapter 1: Appendix 1.1 397

Table 1.2b cont. Reference Species Amount collected Abundance Purpose of Collection/ Fate of organism

Lord, 1986 Nucella lapillus 30 NR Bacterial contamination in intracapsular fluid Amer. Malac. Bull. Buccinum undatum 3 egg capsules Thais haemastoma 4 egg capsules Martel et al., 1986 Buccinum undatum NR Common Egg laying behaviour. Also notes urchin J. Exp. Mar, Biol. Ecol. predation Jannun and Coe, 1987 Murex trunculus 1-2kg NR Bromoperoxidase activity Comp. Biochem. Physiol. Chapman et al., 1988 Polinices incei NR Aust. Response to diesel oil Aust. J. Mar. Freshwater Res. Distribution Rudman and Avern, 1989 Rostanga arbutus 75 Very common Morphology + eggs described Zool. J. Linn. Soc. R. bifurcata 27 Wide dist. R. calumus sp. nov 23 NR R. muscula 15 Common Rostanga bassia sp. nov 3 Restricted dist. Rostanga australis sp. nov 23 Restricted dist. Stöckmann-Bosbach and Nucella lapillus NR NR Biochemical study on egg capsules Althoff, 1989, Marine Biology Rawlings, 1990 Nucella emarginata 300 (all the Wide distribution Egg capsules and predation. Capsules Biol. Bull. reproductively mature spawned in aquaria and preserved snails at 3 sites) Biermann et al., 1992 Archidoris montereyensis 14 NR Radiation and fouling on egg deposition Mar. Ecol. Prog. Ser.

Chapter 1: Appendix 1.1 398

Table 1.2b cont. Reference Species Amount collected Abundance Purpose of Collection/ Fate of organism

Middelfart, 1992a Chicoreus brunneus 5 NR Description of eggs ect. Phuket mar. biol. Cent. Spec. Publ. C. torrefactus 2 NR Middelfart, 1992b C. torrefactus 110 (some from shell NR Description of shell Phuket mar. biol. Cent. Spec. Publ. traders) Havenhand, 1993 Adalaria proxima NR Annual and Model of relationship between egg to Mar. Ecol. Prog. Ser. Onchidoris muricata NR semelparous juvenile period and larval type. The eggs of these two species were used to test the model. I presume they were collected Kohn, 1993 Conus dorreensis 16 egg masses Dense pop. Development and life history Proc. 5th Intern. Marine. Biol. C. klemae 11 capsules Common Workshop C. anemone 4 egg masses + several Common juveniles + distributions Knudsen, 1994 Calyptraea extinctorum 8 adults, 4 brooding NR but Descriptions of egg masses. Material mostly Malacofauna of Hong Kong and Crepidula onyx 17 +, 1 brooding distributions trawled. Some egg masses were deposited Southern China III Siphopatella walshi “Several samples” 2 described in aquaria. brooding Cypraea cf. gracilis 2 egg masses + 2 adults Mentions necrosis Crenvolva renovata Several egg mass C. chinensis Egg mass One large (5L) egg mass (Phalium) was Sinum cf. japonicum 2 adults and egg trawled. This was subsampled and the Phalium cf. bisulcatum One 5L communal egg majority was returned to the place it was mass subsampled + collected. several adults Nassarius festivus 6 adults with egg caps Note: the Phalium egg mass looks similar to Rapana bezoar 10 adults my unidentified (Muricid) egg mass from Trigonostoma scalariformis 3 egg capsules + 1 adult Bellambi Inquisitor sp. 1 NR Inquisitor sp. 2 NR Rawlings, 1994, Evolution Nucella emarginata 195 capsules NR Predation on egg capsules- experimental Booth, 1995, J. Exp. Biol. Polinices sordidus >160 egg masses NR Oxygen transport and embryonic development

Chapter 1: Appendix 1.1 399

Table 1.2b cont. Reference Species Amount collected Abundance Purpose of Collection/ Fate of organism

Rawlings, 1995, Veliger Nucella emarginata NR but laboratory raised Lab. Raised Observations on encapsulated development – rearing outside capsules in sterile conditions. Bai et al., 1996 Biomphalaria glabrata NR NR Phenoloxidase activity in eggs Comp. Biochem. Physiol. Cohen and Strathmann, 1996 Melanochlamys diomedea NR NR Oxygen in egg masses Biol. Bull. Haminaea vesicula NR NR Middelfart, 1996 Chicoreus capucinus Several NR Description of egg mass Phuket Mar. Biol. Cent. Spec. Publ. Thais keineri 8 NR Cymia sacellum 533 egg capsules NR Thais tissoti 120 adults + 1 egg mass NR (washed ashore) Thais sp. 2 adults + 1 egg mass Very common Cymia lacera ? (from fish market) NR Drupella rugosa 13 NR Morula granulata few? NR Ergalatax margariticola NR NR Rawlings, 1996 Nucella emarginata NR Abundant UV on egg capsules Mar. Ecol. Prog. Ser. (some NR labraised) N. lamellosa NR NR N. canaliculata NR egg capsules Cumming, 1997 Crenavolva stiatula 7 specimens NR Observations and feeding ecology. Notes The Marine Flora and Fauna of Hong C. cuspis 47 necrosis from egg masses. Kept in aquaria Kong and Southern China IV C. platysia 11 for two weeks. Some died. Others Sandalia rhodia 1 returned?? Phenacovolva subreflexa 1 P. rosea 23 P. brevirostris 8

Chapter 1: Appendix 1.1 400

Table 1.2b cont. Reference Species Amount collected Abundance Purpose of Collection/ Fate of organism

Knudsen, 1997a Crenavolva platysia 1 adult + 1 egg mass NR distribution Description of egg mass:- all material The Marine Flora and Fauna of Hong with 60 capsules reported deposited in museum Kong and Southern China IV Crenavolva striatula As above Phenacovolva rosea 1 adult + 1 egg mass with 21 capsules Prionovolva nivea 1 adult and numerous egg capsules from two gorgonian specimens Nassarius siquijorensis 30 egg capsules + several hundred laid in aquaria Knudsen, 1997b Calyptraea extinctorium 20 specimens Widely Observations on morphology and The Marine Flora and Fauna of Hong distributed development. Specimens preserved in Kong and Southern China IV museum Knudsen, 1997c Nerita histrio 6 Found often Observations on adults and egg masses The Marine Flora and Fauna of N. hostrio egg capsules 4 Wide dist. Darwin Harbour Nerita reticulata 17 Small colony Pugilina cochlidium eggs NR Few specimens Nassarius dorsatus 26 specimens Abundant Nassarius fraudator 34 specimens Abundant Cronia margariticola eggs 2 egg capsules Very common

NOTE

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UNIVERSITY OF WOLLONGONG

Chapter 2: Appendix 2.1 401

Chapter 2: Appendix 2

Appendix 2.1

Species lists of molluscs at 13 intertidal reefs on the Wollongong Coast, NSW, Australia.

The species of molluscs recorded at 13 intertidal reefs along the Wollongong Coast, NSW Australia are listed, for both a single inventory and the accumulated records from 1995-1998. A description of the habitat and the sampling dates are also provided for each site.

The occurrence of each species refers to it’s estimated relative abundance at that site (refer to Methods, Section 2.3.2).

Records of egg masses and shells observed amongst beach debris are also included for each site.

The molluscs recorded in previous studies at the site are included where relevant.

Appendix 2.1a: Austinmer Appendix 2.1b: Bass Point Appendix 2.1c: Bellambi Appendix 2.1d: Bulli Appendix 2.1e: Coalcliff Appendix 2.1f: Coledale Appendix 2.1g: Flagstaff Hill Appendix 2.1h: North Shellharbour Appendix 2.1i: North Wollongong Appendix 2.1j: Scarborough Appendix 2.1k: South Shellharbour Appendix 2.1l: Towradgi Appendix 2.1m: Wombarra Chapter 2 Appendix 2.1 402

Appendix 2.1a: Austinmer Location: North and south ends of Austinmer Beach. Habitat: On the south-end of Austinmer Beach is a wave exposed rock platform with a steep drop-off. There is a stormwater drain and three artificial swimming pools (refer to Appendix 2.2a) on this rock platform. East of the large swimming pool is a boulder- filled hollow that can only be accessed on very low tides. The boulders are all fixed by the growth of encrusting algae. There is also a small reef flat with Pyura stolonifera and large patches of Caulerpa filiformis north east of the pools. On the northern end of Austinmer Beach there is a large wave exposed rock platform with a steep drop-off. There is a channel and several crevices running through this rock platform, some of which are dominated by Caulerpa filiformis. This platform becomes periodically inundated with sand. Inventory: 14/12/97, Tide = 0.2 Other sampling dates: 4/4/95; 10/4/95; 27/4/95; 15/5/95; 19/5/95; 20/5/95 26/7/95; 19/9/95; 26/7/96; 31/11/96; 7/6/97; 8/6/97; 16/11/97; 8/1/98; ¾/98; 8/5/98; 26/8/98. Total number of sampling periods: 18 Family Species Occurrence Inventory Eggs Shells Neritidae Nerita atramentosa Abundant ✔ ✔ Common Patellidae Patella peronii Abundant ✔ Common Patellidae Patella chapmani - ✖ Common Patellidae Cellana tramoserica Abundant ✔ Common Acmaeidae Notoacmea petterdi Common ✔ Acmaeidae Patelloida alticostata Common ✔ Acmaeidae Patelloida latistrigata Uncommon ✖ Uncommon Acmaeidae Patelloida cf. mufria Uncommon ✔ Haliotidae Haliotis ruber - ✖ Uncommon Fissurellidae Scutus antipodes Uncommon ✔ Uncommon Fissurellidae Amblychilepas nigrita Rare ✖ Fissurellidae Diodora lineata Rare ✖ Fissurellidae Montfortula rugosa Abundant ✔ Common Trochidae Austrocochlea constricta Abundant ✔ Uncommon Trochidae Cantharidella picturata Common ✔ Uncommon Trochidae Phasianotrochus eximius Uncommon ✔ Uncommon Turbinidae Turbo undulatus Common ✔ Common Littorinidae Littorina acutispira Common ✔ Littorinidae Littorina unifasciata Abundant ✔ Littorinidae Nodilittorina pyramidalis Abundant ✖ Littorinidae Bembicium nanum Abundant ✔ ✔ Common Ranellidae Cabestana spengleri Uncommon ✖ Uncommon Muricidae Morula marginalba Abundant ✔ Common Muricidae Dicathais orbita Common ✔ ✔ Common Columbellidae Mitrella sp. Common ✔ Siphonariidae Siphonaria denticulata Abundant ✔ ✔ Uncommon Siphonariidae Siphonaria funiculata Abundant ✔ Uncommon Siphonariidae Siphonaria zelandica Common ✖ ✔ Oxynoidae Oxynoe viridis Rare (1) ✖ Dorididae Rostanga bassia Rare (3) ✔ Ischnochitonidae Ischnochiton australis Uncommon ✔ Ischnochitonidae Ischnochiton versicolour Uncommon ✔ Ischnochitonidae Ischnochiton elongatus Uncommon ✔ Chitonidae Onithochiton quercinus Common ✔ Chitonidae Chiton pelliserpentis Common ✔ Chitonidae Rhyssoplax jugosus Rare ✖

Chapter 2 Appendix 2.1 403

Appendix 2.1a Austinmer continued. Family Species Occurrence Inventory Eggs Shells Mopalidae Plaxiphora albida Common ✔ Mytilidae Xenostrobus pulex Abundant ✔ Ostreidae Saccostrea glomerata Common ✔ Octopodidae Octopus tetricus Rare ✖ Total number of species 38 29 5 181

1 Two species were only recorded from the presence of shells Chapter 2 Appendix 2.1 404

Appendix 2.1b: Bass Point Location: On the northern side of Bass Pint, approximately half way between the entrance gates and the point. Habitat: This is a moderately exposed north facing rock platform with a number of large, boulder-filled pools and channels. Some of the pools are high on the rock platform and therefore sheltered from strong swell. The area is unusually complex and represents the largest uninterrupted rock platform in the Wollongong region. Inventory: 16/11/97, Tide = 0.2 Other sampling dates: 16/10/97; 27/4/98 Total number of sampling periods: 3 Family Species Occurrence Inventory Previous Eggs Shells studies2 Architectonicidae Philippia lutea Rare (1) ✔ ! Neritidae Nerita atramentosa Abundant ✔ ✔ ✔ Common Patellidae Patella peronii Abundant ✔ ✔ Common Patellidae Cellana tramoserica Abundant ✔ ✔ Common Acmaeidae Notoacmea petterdi Abundant ✔ ! Common Acmaeidae Patelloida alticostata Common ✔ ! Common Acmaeidae Patelloida cf. mufria Uncommon ✔ ! Uncommon Haliotidae Haliotis ruber Rare (1) ! ! Rare Haliotidae Haliotis coccoradiata - ! ! Rare (1) Fissurellidae Scutus antipodes Common ✔ ! Uncommon Fissurellidae Amblychilepas nigrita Uncommon ✔ ! Fissurellidae Diodora lineata Uncommon ✔ ! Fissurellidae Montfortula rugosa Abundant ✔ ! Common Trochidae Austrocohlea constricta Abundant ✔ ✔ Common Trochidae Cantharidella picturata Abundant ✔ ! Trochidae Clanculus brunneus Common ✔ ! Trochidae Clanculus floridus Rare (1) ! ! Trochidae Granata imbricata Common ✔ ! Uncommon Trochidae Phasianotrochus eximius Common ✔ ! Uncommon Trochidae Stomatella impertusa Common ✔ ! Trochidae Tallorbis roseolus Uncommon ✔ ! Uncommon Turbinidae Astralium rhodostomum Rare (4) ✔ ! Turbinidae Astralium squamiferum Uncommon ! ! Turbinidae Astralium tentoriformis Abundant ✔ ✔ Common Turbinidae Turbo undulatus Abundant ✔ ✔ Common Turbinidae Turbo torquatus - !3 ! Rare Cerithiidae Cacozeliana granaria Common ✔ ! Common Planaxidae Hinea brasiliana Common ✔ ! Uncommon Littorinidae Bembicium nanum Abundant ✔ ✔ ✔ Common Littorinidae Littorina acutispira Abundant ✔ ! Littorinidae Littorina unifasciata Abundant ✔ ✔ Littorinidae Nodilittorina pyramidalis Abundant ✔ ! Rissoidae Rissoina sp. Abundant ✔ ! Common Rissoidae Rissoina (Rissolina) sp. Common ✔ ! Uncommon Cypraeidae Cypraea caputserpentis - ! ! Rare (1) Cypraeidae Cypraea sp. 4 Rare (4) ! ! Ranellidae Cabestana spenglerii Common ✔ ✔ Uncommon Ranellidae Charonia lampas Uncommon ✔ !

2 Marine Pollution Research Pty Ltd. 1995. Ecological survey and assessment of marine ecological impacts. Appendix 11 Environmental Impact Statement: Shell Cove Boatharbour/Marina. Lester Firth (aust) Pty Ltd. Edgecliff, NSW, Australia. 3 One shell was found 4 juveniles – brown shell, 4-5cm Chapter 2 Appendix 2.1 405

Appendix 2.1b: Bass Point continued. Family Species Occurrence Inventory Previous Eggs Shells studies5 Ranellidae Cymatium parthenopeum Rare (1) ! ! Ranellidae Ranella australasia Rare (2) ! ! Rare Ranellidae Sassia parkinsonia Rare (4) ✔ ! Rare Epitoniidae Epitonium sp. - ! ! Uncommon Epitoniidae Opalia australis - !6 ! Rare Muricidae Agnewia tritoniformis Common ✔ ! ✔ Common Muricidae Bedeva hanleyi Uncommon ✔7 ! ✔ Uncommon Muricidae Dicathais orbita Common ✔ ! Common Muricidae Morula marginalba Abundant ✔ ✔ ✔ Common Buccinidae Engina australis Uncommon ✔ ! Mitridae Mitra boudi Uncommon ✔4 ! ✔ Rare Mitridae Mitra carbonaria Uncommon ✔4 ! ✔ Mitridae Mitra glabra - ! ! Rare (1) Conidae Conus paperliferus Uncommon ✔4 ! ✔ Rare Columbellidae Mitrella sp. Common ✔ ! Marginellidae Volvarina mustelina Common ✔ ! Siphonariidae Siphonaria denticulata Abundant ✔ ! ✔ Common Siphonariidae Siphonaria funiculata Abundant ✔ ! Common Aplysiidae Aplysia dactylomela Rare (1) ! ✔ ✔ Aplysiidae Aplysia sydneyensis Rare (1) ! ! Aplysiidae Dolabrifera dolabrifera8 Common ✔ ! ✔ Pleurobranchidae Berthellina citrina Rare (5) ✔9 ! ✔ Pleurobranchidae Pleurobranchus peroni Rare (1) ✔ ! Bullinidae Bullina lineata Rare (3) ✔ ! ✔ Oxynoidae Oxynoe viridis Rare (1) ! ! Dendrodorididae Dendrodoris gemmacea Rare (1) ! ! Dendrodorididae Dendrodoris fumata Uncommon ✔ ✔ ✔ Dendrodorididae Dendrodoris nigra Rare (1) ! ! ✔ Dendrodorididae Doriopsilla sp.10 Rare (1) ! ! Dorididae Jorunna pantherina Rare (4) ! ! ✔ Dorididae Jorunna sp. (purple) Rare (1) ✔ ! Dorididae Rostanga bassia Rare (4) ! ! ✔ Dorididae Glossodoris atromarginator Rare (1) ! ! Chromodorididae Ceratosoma amoena Rare (2) ✔ ! Chromodorididae Hypselodoris bennetti Rare (1) ! ! Chromodorididae Mexichromis mariei Rare (1) ! ! Polyceridae Plocampherus imperialis Rare (3) ✔ ! Glaucidae Austraeolis ornata Rare (3) ✔4 ! ✔ Glaucidae Spurilla sp. Rare (1) ! ! Aeolidiidae Aeolidiella foulisi Common ✔4 ! ✔ Unidentified Opisthobranch sp. 211 Rare (1) ! ! Unidentified Opisthobranch eggs sp 1012 Rare (1) ! ! ✔

5 Marine Pollution Research Pty Ltd. 1995. Ecological survey and assessment of marine ecological impacts. Appendix 11 Environmental Impact Statement: Shell Cove Boatharbour/Marina. Lester Firth (aust) Pty Ltd. Edgecliff, NSW, Australia. 6 Two shells were found 7 Both adults and eggs were found 8 I found a couple of white individuals. I haven’t seen this colour morph at any other site. 9 I also found an egg ribbon that looked like it could be Berthillina, however, several nudibranchs lay egg ribbons that are indistinguishable with that of Berthillina. 10 I think this nudibranch is Doriopsilla carneola but it could be a different species and possibly even a different genus. 11 Black body with long tail and orange rhinophores 12 Large (>20cm diameter) spiralled frilly egg ribbon with yellow eggs Chapter 2 Appendix 2.1 406

Appendix 2.1b: Bass Point continued. Family Species Occurrence Inventory Previous Eggs Shells studies13 Ischnochitonidae Callochiton crocina Rare (3) ✔ ! Ischnochitonidae Ischnochiton australis Abundant ✔ ! Uncommon Ischnochitonidae Ischnochiton elongatus Common ✔ ! Ischnochitonidae Ischnochiton versicolour Abundant ✔ ! Chitonidae Chiton pelliserpentis Common ✔ ! Chitonidae Onithochiton quercinus Common ✔ ! Chitonidae Rhyssoplax jugosus Common ✔ ! Mopalidae Plaxiphora albida Uncommon ✔ ! Acanthochitonidae Crytoplax mystica Common ✔ ! Mytilidae Mytilus edulis Uncommon ✔ ! Uncommon Mytilidae Trichomya hirsuta Common ✔ ! Common Pectinidae Scaeochlamys livida Uncommon ✔ ! Anomiidae Anomia trigonopis - ! ! Uncommon Montacutidae Kellia rotunda Common ✔ ! Ostreidae Saccostrea glomerata Common ✔ ! Octopodidae Octopus sp. Rare (4) ✔ ! Octopodidae Hapalochlaena maculosa Rare (1) ! ! Total Number of Species 8914 6915 11 20 4116

13 Marine Pollution Research Pty Ltd. 1995. Ecological survey and assessment of marine ecological impacts. Appendix 11 Environmental Impact Statement: Shell Cove Boatharbour/Marina. Lester Firth (aust) Pty Ltd. Edgecliff, NSW, Australia. 14 Number of species that were:- Abundant = 23; Common = 21; Uncommon = 15; Rare = 29. One rare species is only recorded from the presence of an egg ribbon. It is possible that this egg ribbon was laid by one of the species of nudibranch that have been recorded in this table. 15 Three of the species recorded in the inventory were only detected by the presence of their egg masses. 16 The shells from seven species were found that were not recorded live. Chapter 2 Appendix 2.1 407

Appendix 2.1c: Bellambi

Location: On the north side of Bellambi Point, in between the artificial swimming pool (refer to Appendix 2.2b) and the boatramp. Habitat: This is a north facing, low relief boulder field, sheltered by Bellambi Point and a breakwater. There are a number of rocky outcrops, as well as, boulder-filled hollows, which retain water on even the lowest tides. This site is close to a sewage outfall (Bellambi Point) and the water does smell extremely unpleasant at times. The introduced species Caulerpa filiformis is dominant in the sublittoral zone and has infiltrated the water retaining hollows in the intertidal zone. Inventory: 1/2/98; Tide = 0.2 Other sampling dates: 22/10/96; 30/10/96; 2/11/96; 9/12/96; 12/12/96; 10/12/96; 23/4/97; 24/4/97; 19/7/97; 3/8/97; 18/8/97; 19/10/97; 29/11/97; 31/12/97; 31/1/98; 28/3/98; 23/4/98; 25/4/98; 27/8/98. Total number of sampling events: 20 Family Species Occurrence Inventory Previous Eggs Shells studies17 Neritidae Nerita atramentosa Abundant ✔ ✔ ✔ Common Patellidae Patella peronii Abundant ✔ ✖ Common Patellidae Patella chapmani - ✖ ✖ Common Patellidae Cellana tramoserica Abundant ✔ ✔ Uncommon Acmaeidae Notoacmea petterdi Common ✔ ✖ Uncommon Acmaeidae Patelloida alticostata Common ✔ ✔ Common Acmaeidae Patelloida cf. mufria Uncommon ✔ ✖ Haliotidae Haliotis ruber Rare (2) ✔ ✖ Haliotidae Haliotis coccoradiata - ✖ ✖ Rare Fissurellidae Scutus antipodes Common ✔ ✖ Uncommon Fissurellidae Amblychilepas nigrita Uncommon ✔ ✖ Fissurellidae Diodora lineata Uncommon ✖ ✖ Fissurellidae Montfortula rugosa Abundant ✔ ✔ Common Trochidae Austrocochlea constricta Abundant ✔ ✔ Common Trochidae Cantharidella picturata Abundant ✔ ✖ ✔ Trochidae Clanculus brunneus Uncommon ✔ ✖ Trochidae Clanculus floridus - ✖ ✖ Rare Trochidae Granata imbricata Abundant ✔ ✖ Rare Trochidae Phasianotrochus eximius Uncommon ✔ ✖ Rare Trochidae Stomatella impertusa Common ✔ ✖ Trochidae Tallorbis roseolus Uncommon ✔ ✖ Rare Turbinidae Astralium tentoriformis Common ✔ ✖ Uncommon Turbinidae Astralium rhodostomum Rare ✔ ✖ Turbinidae Astralium squamiferum Rare ✔ ✖ Turbinidae Turbo undulatus Abundant ✔ ✖ 18 Common Turbinidae Turbo torquatus Rare (1) ✖ ✔ 2 Rare Cerithiidae Cacozeliana granaria Common ✔ Littorinidae Littorina unifasciata Abundant ✔ ✔ Littorinidae Littorina acutispira Abundant ✔ Littorinidae Nodilittorina pyramidalis Common ✔ ✖ Littorinidae Bembicium nanum Abundant ✔ ✔ ✔ Common

17 Miskiewicz and Lock, 1991, Intertidal Communities in Marine Investigations for the Bellambi and Port Kembla Sewage Treatment Plant Upgrading. Volume 2. Biology, Sediments Bioaccumulation and Water Quality. Environment Management Unit, Water Board, NSW, Australia. 18 The large numbers recorded by Miskiewicz and Lock (1991) suggest they may have misnamed Turbo undulatus as T. torquatus Chapter 2: Appendix 2.1c 408

Appendix 2.1c: Bellambi continued. Family Species Occurrence Inventory Previous Eggs Shells studies Rissoidae Rissoina sp. Common ✔ ✖ Common Rissoidae Rissoina (Rissolina) sp. Common ✔ ✖ Uncommon Cypraeidae Cypraea caputserpentis Rare (1) ✖ ✖ Rare (2) Cypraeidae Cypraea vitellus - ✖ ✖ Rare (1) Cypraeidae Cypraea clandestina - ✖ ✖ Rare (1) Cypraeidae Cypraea annulus Rare (1) ✖ ✖ Cypraeidae Cypraea sp. 119 Rare (3) ✔ ✖ Cypraeidae Cypraea sp. 220 Rare (6) ✖ ✖ Cassidae Semicassis labiatum Rare (1) ✔ ✖ Rare (2) Ranellidae Cabestana spengleri Common ✔ ✖ ✔ 21 Uncommon Ranellidae Ranella australasia Uncommon ✔ ✖ Epitoniidae Epitonium sp. Rare (3) ✖ ✖ Uncommon Epitoniidae Opalia australis - ✖ ✖ Rare (1) Epitoniidae Opalia ballinensis - ✖ ✖ Rare (2) Janthinidae Janthina janthina - ✖ ✖ Uncommon Muricidae Morula marginalba Abundant ✔ ✔ ✔ Common Muricidae Dicathais orbita Common ✔ ✔ ✔ 5 Common Muricidae Agnewia tritoniformis Common ✖ ✖ ✔ Uncommon Muricidae Bedeva hanleyi Rare (3) ✖ ✖ Mitridae Mitra carbonaria Uncommon ✖ ✖ ✔ Rare Conidae Conus paperliferus Uncommon ✔22 ✖ ✔ Rare Conidae Conus anemone Rare (1) ✖ ✖ Rare Columbellidae Mitrella sp. Uncommon ✔ ✖ Uncommon Marginellidae Volvarina mustelina Uncommon ✔ ✖ Unidentified Unidentified eggs sp. 223 Rare ✖ ✖ ✔ Unidentified Unidentified eggs sp. 324 Rare ✖ ✖ ✔ Unidentified Unidentified eggs sp. 425 Rare (1) ✖ ✖ ✔ Siphonariidae Siphonaria denticulata Abundant ✔ ✔ ✔ Common Siphonariidae Siphonaria funiculata Abundant ✔ ✖ Common Siphonariidae Siphonaria zelandica Common26 ✖ ✖ ✔ Siphonariidae Siphonaria virgulata - ✖ ✔ Aplysiidae Aplysia sydneyensis Common10 ✖ ✖ ✔ Aplysiidae Aplysia juliana Common10 ✔ ✖ ✔ Aplysiidae Dolabrifera dolabrifera Common ✔ ✖ ✔ Pleurobranchidae Pleurobranchus peroni Rare (2) ✖ ✖ Pleurobranchidae Pleurobranchea sp. Uncommon10 ✖ ✖ ✔ Pleurobranchidae Berthellina citrina Uncommon10 ✖ ✖ ✔ Polybranchiidae Polybranchia orientalis Rare (1) ✖ ✖ Dendrodorididae Dendrodoris fumata Uncommon (8) ✖ ✖ ✔ Dendrodorididae Dendrodoris nigra Rare (4) ✖ ✖ ✔ Dendrodorididae Doriopsilla miniata Rare (1) ✖ ✖ Dendrodorididae Doriopsilla carneola Rare (1) ✖ ✖

19 juveniles - brown shell, 3-6cm 20 juveniles - white shell, 1-2cm, some with yellow stripe. 21 Washed up on the beach only 22 Both adults and egg masses were found 23 Hemispherical flat conjoined capsules with large pinkish eggs 24 Large egg mass found on the underside of a boulder. Egg capsules were cojoined and laid on top of one another. Most of the capsules had hatched or were close to hatching. The capsular fluid was orange with hints of purple. 25 Small white droplet shaped egg capsule with one black egg inside 26 These species have fluctuating populations and were not recorded on some field trips Chapter 2: Appendix 2.1c 409

Appendix 2.1c: Bellambi continued. Family Species Occurrence Inventory Previous Eggs Shells studies Dorididae Discodoris fragilis Rare (4) ✖ ✖ ✔ Dorididae Platyodoris sp. Uncommon (8) ✖ ✖ ✔ Dorididae Jorunna sp. (purple) Rare (1) ✖ ✖ Dorididae Rostanga bassia Uncommon ✔27 ✖ ✔ Goniodorididae Goniodoris sp. Uncommon10 ✖ ✖ ✔ Glaucidae Austraeolis ornata Uncommon ✔11 ✖ ✔ Glaucidae Glaucilla marginata Rare (4) ✖ ✖ Aeolidiidae Aeolidiella foulisi Common10 ✔11 ✖ ✔ Ischnochitonidae Ischnochiton australis Abundant ✔ ✖ Rare Ischnochitonidae Ischnochiton versicolour Abundant ✔ ✖ Ischnochitonidae Ischnochiton elongatus Abundant ✔ ✖ Chitonidae Onithochiton quercinus Uncommon ✔ ✔ 28 Rare Chitonidae Chiton pelliserpentis Common ✔ ✖ Rare Chitonidae Rhyssoplax jugosus Uncommon ✔ ✖ Mopalidae Plaxiphora albida Uncommon ✔ ✖ Acanthochitonidae Crytoplax mystica Common ✔ ✖ Mytilidae Xenostrobus pulex Common ✖ ✖ Mytilidae Trichomya hirsuta Uncommon ✖ 29 ✖ Common Anomiidae Anomia trigonopis - ✖ ✖ Uncommon Montacutidae Kellia rotunda Common ✔ ✖ Pectinidae Scaeochlamys livida Rare (1) ✖ ✖ Ostreidae Saccostrea glomerata Common ✔ ✖ Ostreidae Unidentified sp. 1 Rare (2) ✖ ✖ Sepiidae Sepia sp. - ✖ ✖ Uncommon Octopodidae Octopus sp. Rare (4) ✔ ✖ Octopodidae Hapalochlaena maculosa Rare (2) ✖ ✖ Total number of species 9030 56 13 2731 4432

27 Adults and egg masses were found 28 Miskiewicz and Lock (1991) refer to Onithochiton sp. 29 Shells were recorded 30 Number of species that were:- Abundant = 17; Common = 24; Uncommon = 22; Rare = 27 31 The egg capsules of two species were only found washed up on the beach 32 The shells of 11 species that were not recorded live were found. Chapter 2 Appendix 2.1 410

Appendix 2.1d: Bulli Location: Sandon Point, around Bulli foreshore pool. Habitat: This is an exposed rock platform, which slopes gently into ocean. There is a channel through middle of platform with many boulders but the area does dries out on very low tides. The boulders move around a lot during strong swell and on occasions large areas of the rock platform are smothered by sand. There are several deep natural rock pools on the rock platform and these are all dominated by Caulerpa filiformis. The rock platform is raised on the northern side and drops steeply into the ocean. There are many boulders on the northern and eastern sides below the rock platform but these can only be reached on very calm low tides. These boulders are mostly fixed by the growth of encrusting algae. There are a number of rocky outcrops with Pyura stolonifera on the eastern side of the platform but these are difficult to access. There is a large artificial swimming pool on the southern end of the rock platform (Appendix 2.2c). Inventory: 27/1/98; Tide = 0.2 Other sampling dates: 10/4/95; 18/10/95; 25/7/96; 17/8/96; 10/10/96; 19/11/96; 27/11/96; 5/4/97; 4/7/97; 1/8/97; 19/9/97; 27/10/97; 2/11/97; 17/11/97; 31/1/98, 26/4/98; 13/6/98; 24/8/98. Total number of sampling dates: 18

Family Species Occurrence Inventory Eggs Shells Neritidae Nerita atramentosa Abundant ✔ ✔ Common Patellidae Patella peronii Abundant ✔ Common Patellidae Patella chapmani - ✖ Common Patellidae Cellana tramoserica Abundant ✔ Common Acmaeidae Notoacmea petterdi Common ✔ Uncommon Acmaeidae Patelloida alticostata Common ✔ Uncommon Acmaeidae Patelloida cf. mufria Uncommon ✔ Haliotidae Haliotis ruber Rare (1) ✖ Rare Fissurellidae Scutus antipodes Common ✔ Uncommon Fissurellidae Amblychilepas nigrita Rare ✖ Fissurellidae Diodora lineata Rare (3) ✖ Fissurellidae Tugali sp. Rare (1) ✖ Fissurellidae Montfortula rugosa Abundant ✔ Uncommon Trochidae Austrocochlea constricta Abundant ✔ Common Trochidae Cantharidella picturata Abundant ✔ ✔ Uncommon Trochidae Phasianotrochus eximius Uncommon ✔ Uncommon Trochidae Granata imbricata - ✖ Rare Turbinidae Turbo undulatus Abundant ✔ Common Cerithiidae Cacozeliana granaria Uncommon ✔ Uncommon Littorinidae Littorina acutispira Abundant ✔ Littorinidae Littorina unifasciata Abundant ✔ Littorinidae Nodilittorina pyramidalis Common ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ Common Rissoidae Rissoina sp. Uncommon ✔ Uncommon Rissoidae Rissoina (Rissolina) sp. Abundant ✔ Uncommon Cassidae Semicassis labiatum - ✖ Rare Ranellidae Cabestana spengleri Common ✔ ✔33 Common Ranellidae Ranella australasia - ✖ Rare Janthinidae Janthina janthina - ✖ Common Muricidae Morula marginalba Abundant ✔ Common Muricidae Dicathais orbita Common ✔ ✔ Common Muricidae Lepsiella reticulata Uncommon ✔ Rare

33 Egg masses only recorded from beach debris Chapter 2 Appendix 2.1 411

Appendix 2.1d: Bulli continued. Family Species Occurrence Inventory Eggs Shells Mitridae Mitra sp. - ✖ Rare Conidae Conus sp. - ✖ Uncommon Columbellidae Mitrella cf. semiconvexa Uncommon ✔ Siphonariidae Siphonaria denticulata Abundant ✔ ✔ Uncommon Siphonariidae Siphonaria funiculata Common ✔ Common Siphonariidae Siphonaria zelandica Common34 ✖ ✔ Elysiidae Elysia australis Uncommon2 ✖ ✔ Aplysiidae Aplysia juliana Uncommon2 ✖ ✔ Aplysiidae Dolabrifera dolabrifera Rare (1) ✖ Pleurobranchidae Pleurobranchus peroni Rare (1) ✔ Pleurobranchidae Pleurobranchea sp. Rare35 ✔ ✔ Hydatinidae Hydatina physis Rare (3) ✔ ✔ Chromodorididae Hypselodoris bennetti Rare ✔ ✔ Polyceridae Plocamopherus imperialis Rare (2) ✖ ✔ Dendrodorididae Doriopsilla carneola Rare (2) ✔ Dendrodorididae Doriopsilla miniata Rare (1) ✔ Glaucidae Austraeolis ornata Uncommon ✖ ✔ Aeolidiidae Aeolidiella foulisi Rare ✖ ✔ Dorididae Rostanga bassia Uncommon ✔ Unidentified Unidentified eggs sp. 1136 Rare (1) ✔ ✔ Ischnochitonidae Ischnochiton australis Common ✔ Uncommon Ischnochitonidae Ischnochiton versicolour Common ✔ Ischnochitonidae Ischnochiton elongatus Common ✔ Chitonidae Onithochiton quercinus Uncommon ✔ Uncommon Chitonidae Chiton pelliserpentis Common ✔ Uncommon Chitonidae Rhyssoplax jugosus Rare ✖ Mopalidae Plaxiphora albida Uncommon ✔ Rare Mytilidae Mytilus edulis - ✖ 37 Uncommon Mytilidae Xenostrobus pulex Abundant2 ✔ Anomiidae Anomia trigonopis - ✖ Uncommon Ostreidae Saccostrea glomerata Common ✔ Sepiidae Sepia sp. - ✖ Uncommon Octopodidae Octopus sp. Rare ✖ Total number of species 5538 40 1639 3640

34 These species have fluctuating populations and were not recorded on every field trip. 35 Only the egg ribbons were recorded for this species 36 Small (~2mm) firm gelatinous ribbon with dull creamy apricot eggs 37 The shells of this species were found 38 Number of species that were:- Abundant = 14; Common = 13; Uncommon = 12; Rare = 16 39 The egg masses from 15 species were recorded in the intertidal area. The egg mass from one further species was found washed up on the beach. 40 Ten species were only recorded from the presence of shells Chapter 2 Appendix 2.1 412

Appendix 2.1e: Coalcliff Location: Southern end of Coalcliff Beach Habitat: This is a large wave exposed rock platform with mostly steep drop-offs into the ocean. The northern end of the platform is quite complex, with many crevices and some boulder filled channels. Below the platform on the northern end there is a small boulder field, which can only be accessed on calm low Spring tides. Most of the boulders are firmly embedded in the substrate and/or fixed by encrusting growth. There is an artificial swimming pool on the north-western side of the reef (Appendix 2.2d) Near the pool there are two seepage drains where fresh water spills onto the rock platforms. The impacts of these drains are fairly localised. The introduced species of alga Caulerpa filiformis is common in the sublittoral zone around the northern end of the rock platform has infiltrated the intertidal area in a number of crevices and rock pools. Inventory: 2/1/98; Tide = 0.2 Additional sampling dates: 12/5/95; 23/7/95; 29/11/96; 20/2/97; 28/5/97; 21/8/97; 3/10/97, 28/2/98, 22/6/98 Total number of sampling events: 10 Family Species Occurrence Inventory Eggs Shells Architectonicidae Philippia lutea Rare (1) ✔ Neritidae Nerita atramentosa Abundant ✔ ✔ Common Patellidae Patella peronii Abundant ✔ Common Patellidae Patella chapmani - ✖ Common Patellidae Cellana tramoserica Abundant ✔ Common Acmaeidae Notoacmea petterdi Abundant ✔ Uncommon Acmaeidae Patelloida alticostata Common ✔ Common Acmaeidae Patelloida cf. mufria Uncommon ✖ Haliotidae Haliotis ruber Uncommon ✔ Rare Fissurellidae Scutus antipodes Uncommon ✔ Rare Fissurellidae Amblychilepas nigrita Uncommon ✔ Fissurellidae Diodora lineata Uncommon ✖ Fissurellidae Montfortula rugosa Abundant ✔ Common Trochidae Austrocohlea constricta Abundant ✔ Common Trochidae Cantharidella picturata Abundant ✔ ✔ Uncommon Trochidae Tallorbis roseolus Rare ✔ Rare Trochidae Herpetopoma aspersa Rare (2) ✖ Trochidae Clanculus brunneus Rare (1) ✖ Rare (1) Trochidae Phasianotrochus eximius Uncommon ✖ Uncommon Trochidae Granata imbricata - ✖ Uncommon Turbinidae Turbo torquatus Rare (1) ✖ Rare Turbinidae Turbo undulatus Abundant ✔ Common Littorinidae Littorina acutispira Abundant ✔ Littorinidae Littorina unifasciata Abundant ✔ Littorinidae Nodilittorina pyramidalis Abundant ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ Common Ranellidae Cabestana spengleri Uncommon ✔ ✔41 Common Ranellidae Ranella australasia - ✖ 42 Rare Janthinidae Janthina janthina - ✖ Uncommon Muricidae Morula marginalba Abundant ✔ Common Muricidae Dicathais orbita Common ✔ ✔1 Common Muricidae Agnewia tritoniformis43 Rare (3) ✖ Uncommon Muricidae Bedeva hanleyi Rare ✔ Muricidae Unidentified Muricopsinae44 - ✖ Rare (1)

41 Hatched egg mass washed on beach 42 Shells were found washed up but no live individuals 43 Could also be Lepsiella vinosa. I find these two species difficult to distinguish Chapter 2 Appendix 2.1 413

Appendix 2.1e: Coalcliff continued. Family Species Occurrence Inventory Eggs Shells Buccinidae Engina australis Rare (1) ✖ Mitridae Mitra sp. - ✖ Uncommon Conidae Conus sp. - ✖ Common Columbellidae Mitrella cf. semiconvexa Common ✔ Uncommon Epitoniidae Opalia australis - ✖ 2 Rare (1) Epitoniidae Opalia granosa - ✖ Rare (1) Siphonariidae Siphonaria denticulata Abundant ✔ ✔ Common Siphonariidae Siphonaria funiculata Abundant ✔ Common Aplysiidae Aplysia parvula Rare (2) ✖ Pleurobranchidae Berthellina citrina45 Rare (1) ✔ Dendrodorididae Dendrodoris cf. fumata Rare ✔ Dorididae Rostanga bassia Uncommon ✔ Dorididae Jorunna pantherina Rare (3) ✖ Ischnochitonidae Ischnochiton australis Uncommon ✔ Uncommon Ischnochitonidae Ischnochiton versicolour Uncommon ✔ Ischnochitonidae Ischnochiton elongatus Common ✔ Chitonidae Onithochiton quercinus Uncommon ✔ Uncommon Chitonidae Chiton pelliserpentis Common ✔ Uncommon Chitonidae Rhyssoplax jugosus Rare ✔ Mopalidae Plaxiphora albida Uncommon ✔ Uncommon Mytilidae Trichomya hirsuta - ✖ 2 Common Spondylidae Spondylus cf. tenellus Rare (1) ✖ Anomiidae Anomia trigonopis - ✖ Uncommon Ostreidae Saccostrea glomerata Abundant ✔ Sepiidae Sepia sp. - ✖ Uncommon Octopodidae Octopus sp. Rare ✖ Total number of species 4846 3547 648 3949

44 This shell hasn’t been identified by the Museum but it could be Murexiella phantom or Muricopsis purpurispina. 45 juvenile ~1mm long 46 Number of species that were:- Abundant = 16; Common = 5; Uncommon = 12; Rare = 15 47 Thirty five live species were recorded and the shells of a further three species. 48 The egg masses of 4 species were recorded in the intertidal area and the egg capsules of a further two species were found washed up on the beach. 49 The shells from 12 species were found that were not recorded live. Chapter 2 Appendix 2.1 414

Appendix 2.1f: Coledale Location: Around Coledale swimming pool. North of Coledale caravan park. Habitat: The northern side of this reef is gently sloping, with boulders and crevices in the low intertidal area. In the higher intertidal area the rock platform on retains water in shallow depressions and crevices. Towards the south, the reef opens up into a large exposed rock platform with steep drop-offs. There is one artificial swimming pool on the platform (refer to Appendix 2.2e). South of the pool there are a couple of channels and a small bay with some loose boulders. This area is relatively protected from strong swell. Further south, the reef extends into another large rock platform with steep drop- offs. The introduced species of alga, Caulerpa filiformis is fairly abundant in the sublittoral zone and has extended into the intertidal region in the shallow bay and along crevices. Inventory: 3/1/98, Tide = 0.2 Other sampling dates: 10/5/95; 2/10/95; 31/11/96; 23/2/97; 5/3/97; 15/2/98; 22/2/98; 26/3/98 Total number of sampling events: 9

Family Species Occurrence Inventory Eggs Shells Neritidae Nerita atramentosa Abundant ✔ ✔ Common Patellidae Patella peronii Abundant ✔ Common Patellidae Patella chapmani Rare (6) ✖ Common Patellidae Cellana tramoserica Abundant ✔ Common Acmaeidae Notoacmea petterdi 50 Abundant ✔ Common Acmaeidae Patelloida alticostata Common ✔ Common Acmaeidae Patelloida cf. mufria Uncommon ✖ Fissurellidae Scutus antipodes Uncommon ✔ Rare Fissurellidae Amblychilepas nigrita Uncommon ✔ Fissurellidae Diodora lineata Uncommon ✔ Fissurellidae Montfortula rugosa Abundant ✔ Common Trochidae Austrocohlea constricta Abundant ✔ Common Trochidae Cantharidella picturata Abundant ✔ ✔ Trochidae Herpetopoma aspersa Rare (1) ✖ Trochidae Tallorbis roseolus Rare (1) ✖ Rare Trochidae Phasianotrochus eximius Uncommon ✔ Uncommon Turbinidae Turbo undulatus Abundant ✔ Common Littorinidae Littorina acutispira Abundant ✔ Littorinidae Littorina unifasciata Abundant ✔ Littorinidae Nodilittorina pyramidalis Common ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ Common Rissoidae Rissoina sp. Rare (1) ✔ Rissoidae Rissoina (Rissolina) sp. Uncommon ✔ Ranellidae Cabestana spengleri Uncommon ✔ Common Ranellidae Cymatium parthenopeum Rare (1) ✖ Muricidae Morula marginalba Abundant ✔ ✔ Common Muricidae Dicathais orbita Common ✔ ✔ Common Muricidae Agnewia tritoniformis51 - ✖ Rare (1) Muricidae Ergalatax contracta Rare (4) ✔ ✔ Muricidae Bedeva hanleyi Rare (3) ✖ ✔ Muricidae Lepsiella reticulata Rare (1) ✖ Rare Mitridae Mitra sp. - ✖ Uncommon

50 Some of the small limpets looked identical to Notoacmea mayi but this species is not supposed to occur in NSW. 51 Could also be shells of Lepsiella vinosa Chapter 2 Appendix 2.1 415

Appendix 2.1f: Coledale continued. Family Species Occurrence Inventory Eggs Shells Conidae Conus anemone - ✖ Common Conidae Conus paperliferus - ✖ Rare Columbellidae Mitrella cf. semiconvexa Common ✔ Siphonariidae Siphonaria denticulata Abundant ✔ ✔ Uncommon Siphonariidae Siphonaria funiculata Common ✔ Common Siphonariidae Siphonaria zelandica Common52 ✖ ✔ Pleurobranchidae Berthellina citrina Rare (1)53 ✖ Elysiidae Elysia australis Uncommon3 ✖ ✔ Dendrodorididae Doriopsilla cf. carneola Rare (1) ✖ Dorididae Rostanga bassia Rare (3) ✔ Dorididae Jorunna pantherina Rare (3) ✖ Dorididae Jorunna sp. (purple) Rare (1) ✖ Unidentified Unidentified eggs sp. 1154 Rare (1) ✔ ✔ Ischnochitonidae Ischnochiton australis Abundant ✔ Uncommon Ischnochitonidae Ischnochiton versicolour Uncommon ✔ Ischnochitonidae Ischnochiton elongatus Uncommon ✔ Chitonidae Onithochiton quercinus Common ✔ Chitonidae Chiton pelliserpentis Common ✔ Chitonidae Rhyssoplax jugosus Rare ✔ Mopalidae Plaxiphora albida Uncommon ✔ Mytilidae Xenostrobus pulex Common ✔ Mytilidae Trichomya hirsuta - ✖ 55 Uncommon Anomiidae Anomia trigonopis - ✖ Rare Montacutidae Kellia rotunda Rare (5) ✖ Ostreidae Saccostrea glomerata Common ✔ Octopodidae Octopus sp. Rare ✔ Octopodidae Octopus egg56 - ✖ ✔ Sepiidae Sepia sp. - ✖ Uncommon Sepiidae Sepioteuthis australis57 - ✖ ✔ Total number of species 5258 3859 1360 2761

52 The populations of these species fluctuate over time. The species were not recorded on every trip. 53 One small apricot juvenile 54 Small (~ 5mm) firm, clear, gelatinous ribbon with large white eggs found under boulder. 55 No live specimens were recorded but several shells were found. 56 One egg capsule was found washed up on beach after a storm. This is possibly from a subtidal species. 57 One egg mass was found washed up on beach after storm. 58 Number of species that were:- Abundant = 14; Common = 10; Uncommon = 11; Rare = 17 Two common species and one uncommon species had fluctuating populations and were not recorded on every trip. One rare species was only recorded from the presence of an egg mass. 59 Thirty eight species were recorded live and the shell from one additional species was found. 60 The egg masses from 11 species were found in the intertidal area and the egg masses of a further two species were found washed up on the beach. 61 Seven species were only recorded from shells washed up on the beach. Chapter 2 Appendix 2.1 416

Appendix 2.1g: Flagstaff Hill

Location: Northern end of Wollongong South Beach Habitat: This is an exposed rock platform primarily with steep drop-off but includes a small area of reef flat on the northern side, with extensive Pyura stolonifera beds. This reef flat is separated from the main platform by a small channel and a number of large boulders dominated with barnacles and a narrow Galeolaria zone. The reef flat can only be accessed during very low tides with calm swell conditions. A number of species of molluscs were only observed on this reef flat and not on the rest of the platform. There are a number of natural rock pools along the eastern side of the platform and there is also a shallow artificial swimming pool, which was not sampled. Inventory: 27/2/98, Tide = 0.1 Other sampling dates: 11/4/95; 8/9/95; 12/10/95; 17/10/95; 19/9/96; 11/10/96; 15/10/96; 4/4/97; 24/11/97; 28/11/97; 17/12/97; 6/1/98; 13/1/98; 22/1/98; 19/2/98; 24/4/98 Total number of sampling events: 17 Family Species Occurrence Inventory Previous Eggs Shells studies62 Neritidae Nerita atramentosa Abundant ✔ ✔ ✔ Common Patellidae Patella peronii Common ✔ ✔ Common Patellidae Cellana tramoserica Common ✔ ✔ Common Acmaeidae Notoacmea petterdi Common ✔ ✔ Common Acmaeidae Patelloida alticostata -63 ✖ ✔ Acmaeidae Patelloida latistrigata -64 ✖ ✔ Acmaeidae Patelloida cf. mufria Rare (3) ✔ ✔ Haliotidae Haliotis ruber Rare (1)65 ✖ ✔ Fissurellidae Scutus antipodes Uncommon ✔ ✔ Rare Fissurellidae Amblychilepas nigrita Rare (2)4 ✖ ✖ Fissurellidae Diodora lineata Rare (6) 4 ✔ ✖ Fissurellidae Montfortula rugosa Abundant ✔ ✔ Uncommon Trochidae Austrocohlea constricta Abundant ✔ ✔ Uncommon Trochidae Cantharidella picturata Uncommon ✔ ✖ Trochoidea Tallorbis roseolus - ✖ ✖ Rare Trochidae Phasianotrochus eximius - ✖ ✖ Rare Turbinidae Turbo undulatus Uncommon ✔ ✔ Uncommon Turbinidae Astralium tentoriformis Uncommon4 ✖ ✔66 Rare Littorinidae Littorina acutispira Abundant ✔ ✔ Littorinidae Liittorina unifasciata Abundant ✔ ✔ Littorinidae Nodilittorina pyramidalis Abundant ✔ ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ ✔ Common Ranellidae Charonia lampas - ✖ ✔ Ranellidae Cabestana spengleri Common4 ✔ ✖ ✔67 Uncommon

62 Minchinton, T. E. 1996. Rocky intertidal shores. In Ecological study of Wollongong Harbour, North Wollongong Beach, Fairy Creek and Lagoon, and Stuart and J. P. Galvin Parks. Minchinton, T.E. and Whelan, R.J. Australian Flora and Fauna Research Centre, University of Wollongong. This study presents one species list for the Wollongong intertidal area. I have split the area into three study sites (Flagstaff Hill, North Wollongong Reef and Wollongong Channel). It is possible that some of the species listed by Minchinton (1996) were not recorded on the Flagstaff Hill rock platform and reef. 63 I didn’t record this species at Flagstaff Hill. It is possible that it was recorded from a different site in the Wollongong area by Minchinton and Whelan (1996). 64 It is possible this species occurs here 65 Only recorded on the reef flat 66 Called Australium tentoriforme by Minchinton and Whelan (1996) 67 Hatched egg mass washed up on beach Chapter 2 Appendix 2.1 417

Appendix 2.1g: Flagstaff Hill continued. Family Species Occurrence Inventory Previous Eggs Shells studies68 Muricidae Morula marginalba Abundant ✔ ✔ Uncommon Muricidae Dicathais orbita Common ✔ ✔ ✔ Uncommon Columbellidae Mitrella cf. semiconvexa Uncommon 4 ✔ ✖ Siphonariidae Siphonaria denticulata Abundant ✔ ✔ ✔ Siphonariidae Siphonaria funiculata Abundant ✔ ✖ Siphonariidae Siphonaria virgulata - ✖ ✔69 Siphonariidae Siphonaria sp. - ✖ ✔70 Aplysiidae Aplysia sydneyensis Rare (5) 4 ✖ ✖ Umbraculidae Umbraculum sp. Rare (1) 4 ✔ ✖ Ischnochitonidae Ischnochiton australis Uncommon4 ✔ ✖ Ischnochitonidae Ischnochiton versicolour Uncommon4 ✔ ✖ Ischnochitonidae Ischnochiton elongatus Uncommon4 ✔ ✖ Chitonidae Onithochiton quercinus Uncommon ✔ ✔ Chitonidae Chiton pelliserpentis Common ✔ ✔ Mopalidae Plaxiphora albida Uncommon ✔ ✖ Mytilidae Xenostrobus pulex Common ✖ ✔ Mytilidae Trichomya hirsuta Uncommon4 ✔ ✖ Rare Montacutidae Kellia rotunda Rare (2) 4 ✖ ✖ Ostreidae Saccostrea glomerata Common ✔ ✔71 Total number of species 3672 29 26 573 1674

68 Minchinton, T. E. 1996. Rocky intertidal shores. In Ecological study of Wollongong Harbour, North Wollongong Beach, Fairy Creek and Lagoon, and Stuart and J. P. Galvin Parks. Minchinton, T.E. and Whelan, R.J. Australian Flora and Fauna Research Centre, University of Wollongong. This study presents one species list for the Wollongong intertidal area. I have split the area into three study sites (Flagstaff Hill, North Wollongong Reef and Wollongong Channel). It is possible that some of the species listed by Minchinton (1996) were not recorded on the Flagstaff Hill rock platform and reef. 69 I have not observed this species in the Wollongong region. The species recorded by Minchinton and Whelan (1996) is most likely S. funiculata. 70 Species not identified by Minchinton and Whelan (1996) but could refer to S. zelandica, which is the only other Siphonariid likely to be found in the Wollongong Region 71 This species was recorded by Minchinton and Whelan (1996) as S. commercialis but almost definitely refers to S. glomerata 72 Number of species that were:- Abundant = 10; Common = 8; Uncommon = 11; Rare = 7 73 The egg masses of four species were found in the intertidal area and the egg mass of one species was found washed up on the beach. 74 Not many shells were found washed up on Flagstaff reef or rock platform and I rarely walked along Wollongong South Beach. Chapter 2 Appendix 2.1 418

Appendix 2.1h: North Shellharbour Location: Northern end of Shellharbour South Beach, south of the pool. Habitat: This is a sheltered low relief reef with rocky outcrops, channels and boulder filled pools. The site is facing east but being within the Bass Point embayment it is still significantly sheltered from strong wave action. This reef is potentially threatened by indirect impacts from the approved development of a 350-berth boatharbour/marina. Inventory: 1/3/98; Tide = 0.3 Other sampling dates: 12/1/98 Total number of sampling events: 2 Family Species Occurrence Inventory Previous Eggs Shells studies75 Neritidae Nerita atramentosa Abundant ✔ ✔ ✔ Common Patellidae Patella peronii Abundant ✔ ✔ Common Patellidae Cellana tramoserica Abundant ✔ ✔ Common Patellidae Unidentified sp. A - - ✔ Patellidae Unidentified sp. B - - ✔ Acmaeidae Notoacmea petterdi Abundant ✔ ! Common Acmaeidae Patelloida alticostata Common ✔ ! Uncommon Acmaeidae Patelloida cf. mufria Uncommon ✔ ! Rare Haliotidae Haliotis ruber - ! ! Rare Fissurellidae Scutus antipodes Common ✔ ! Uncommon Fissurellidae Amblychilepas nigrita Uncommon ✔ ! Fissurellidae Diodora lineata Uncommon ✔ ! Fissurellidae Montfortula rugosa Abundant ✔ ✔76 Common Trochidae Austrocohlea constricta Abundant ✔ ✔ Common Trochidae Cantharidella picturata Abundant ✔ ! Uncommon Trochidae Clanculus brunneus Common ✔ ! Trochidae Granata imbricata Common ✔ ! Uncommon Trochidae Phasianotrochus eximius Common ✔ ! Uncommon Trochidae Stomatella impertusa Common ✔ ! Trochidae Tallorbis roseolus Common ✔ ! Rare Turbinidae Astralium rhodostomum Rare (6) ✔ ! Turbinidae Astralium squamiferum Common ✔ ! Turbinidae Astralium tentoriformis Abundant ✔ ! Uncommon Turbinidae Turbo undulatus Abundant ✔ ✔ Common Turbinidae Turbo torquatus - !77 ! Rare Cerithiidae Cacozeliana granaria Abundant ✔ ! Uncommon Dialidae Diala sp. - ! ! Rare (1) Planaxidae Hinea brasiliana Common ! ! Rare Littorinidae Bembicium nanum Abundant ✔ ✔ ✔ Common Littorinidae Littorina acutispira Abundant ✔ ! Littorinidae Littorina unifasciata Abundant ✔ ✔ Littorinidae Nodilittorina pyramidalis Abundant ✔ ✔ Rissoidae Rissoina sp. Abundant ✔ ! Common Rissoidae Rissoina (Rissolina) sp. Common ✔ ! Common Cypraeidae Cypraea caputserpentis - !3 ! Rare (1) Cassidae Semicassis labiatum - ! ! Rare (1) Ranellidae Cabestana spenglerii Common ✔ ! ✔78 Uncommon Ranellidae Charonia lampas Rare (2) ! !

75 Marine Pollution Research Pty Ltd. 1995. Ecological survey and assessment of marine ecological impacts. Appendix 11 Environmental Impact Statement: Shell Cove Boatharbour/Marina. Lester Firth (aust) Pty Ltd. Edgecliff, NSW, Australia. This site was called Shellharbour North by Marine Pollution Research, 1995. 76 Called Montfortula conoidae by Marine Pollution Research (1995). 77 One shell was found but no live specimens. 78 Hatched egg capsules were found washed up Chapter 2 Appendix 2.1 419

Appendix 2.1i: Shellharbour North continued. Family Species Occurrence Inventory Previous Eggs Shells studies79 Ranellidae Ranella australasia Rare (3) ✔ ! Ranellidae Sassia parkinsonia Uncommon ✔ ! Rare Epitoniidae Epitonium sp. Rare (5) ✔ ! Rare Epitoniidae Opalia australis - ! ! Rare (1) Muricidae Agnewia tritoniformis Uncommon ✔ ! Rare Muricidae Bedeva hanleyi Uncommon ✔ ! ✔ Muricidae Dicathais orbita Common ✔ ! ✔4 Uncommon Muricidae Lepsiella reticulata Uncommon ! ! Rare Muricidae Morula marginalba Abundant ✔ ✔ Common Buccinidae Cominella eburnea - !3 ! Rare (1) Buccinidae Engina australis Uncommon ✔ ! Buccinidae Fractolatirus normalis - ! ! Rare (1) Mitridae Mitra sp. - ! ! Rare Conidae Conus anemone - ! ! Rare Conidae Conus paperliferus Rare (5) ✔ ! Rare Columbellidae Euplica versicolour - ! ! Rare (1) Columbellidae Mitrella cf. semiconvexa Common ✔ ! Marginellidae Volvarina mustelina Common ✔ ! Unidentified Unidentified eggs sp. 280 Uncommon ! ! ✔ Siphonariidae Siphonaria denticulata Abundant ✔ ! ✔ Siphonariidae Siphonaria funiculata Abundant ✔ ! Aplysiidae Aplysia parvula Uncommon ✔ ! ✔ Aplysiidae Dolabrifera dolabrifera Uncommon ✔81 ! ✔ Bullinidae Bullina lineata Rare (1) ✔ ! Dendrodorididae Doriopsilla miniata Uncommon ✔6 ! ✔ Dorididae Discodoris fragilis Rare (1) ✔6 ! ✔ Dorididae Jorunna pantherina Rare (2) ✔ ! Dorididae Rostanga bassia Rare (2) ✔ ! Chromodorididae Ceratosoma amoena Rare (1) ✔ ! Glaucidae Austraeolis ornata Rare (1) ! ! Aeolidiidae Aeolidiella foulisi Common ✔6 ! Ischnochitonidae Ischnochiton australis Abundant ✔ ! Ischnochitonidae Ischnochiton elongatus Common ✔ ! Ischnochitonidae Ischnochiton versicolour Abundant ✔ ! Chitonidae Chiton pelliserpentis Common ✔ ✔ Chitonidae Onithochiton quercinus Uncommon ✔ ✔ Uncommon Chitonidae Rhyssoplax jugosus Uncommon ✔ ! Acanthochitonidae Crytoplax mystica Uncommon ✔ ! Mytilidae Trichomya hirsuta Rare (1) ✔ ! Common Montacutidae Kellia rotunda Common ✔ ! Ostreidae Saccostrea glomerata Common ✔ ! Unidentified Bivalve sp. 182 Common ✔ ! Octopodidae Hapalochlaena maculosa Rare (1) ✔ ! Octopodidae Octopus sp. Rare (2) ✔ ! Total Number of Species 6983 6484 14 11 4185

79 Marine Pollution Research Pty Ltd. 1995. Ecological survey and assessment of marine ecological impacts. Appendix 11 Environmental Impact Statement: Shell Cove Boatharbour/Marina. Lester Firth (aust) Pty Ltd. Edgecliff, NSW, Australia. This site was called Shellharbour North by Marine Pollution Research, 1995. 80 Flattened circular capsules with large pinkish eggs 81 Both egg masses and adults were found 82 Almost circular white ridged shell attached to the underside of boulders in clusters 83 Number of species that were:- Abundant = 20; Common = 20; Uncommon = 15; Rare = 14 84 Sixty four species were recorded live and the shells of a further four species were found washed up 85 Only the shells and no live specimens were recorded for 11 species. Chapter 2 Appendix 2.1 420

Appendix 2.1i: North Wollongong Location: Southern end of Wollongong North Beach and north of Wollongong Harbour, around Wollongong Baths. Habitat: This is a fairly large rock platform with both a gently sloping area and areas that drop steeply into the ocean. The reef is facing north-east and is semi- sheltered by the breakwaters of Wollongong Harbour. There are several channels that contain boulders. There are two artificial swimming pools (refer to Appendix 2.2g) and on the ocean-side of the pools there is a patch of boulders, which can only be sampled on low Spring tides in calm conditions. There are a couple of storm water pipes that drain fresh water onto the reef. Inventory: 30/1/98; Tide = 0.1 Other sampling dates: 22/9/95; 19/10/95; 23/7/96; 8/9/96; 17/9/97; 19/9/96; 22/9/96; 18/10/96; 30/1/97; 7/3/97; 5/10/97; 24/11/97; 24/4/98 Total number of sampling periods: 14 Family Species Occurrence Inventory Previous Eggs Shells studies86 Architectonicidae Philippia lutea Rare (1) ✔ ✖ Neritidae Nerita atramentosa Abundant ✔ ✔ ✔ Common Patellidae Patella peronii Abundant ✔ ✔ Common Patellidae Patella chapmani - ✖ ✖ Common Patellidae Cellana tramoserica Abundant ✔ ✔ Common Acmaeidae Notoacmea petterdi Common ✔ ✔ Uncommon Acmaeidae Patelloida alticostata Abundant ✔ ✔ Common Acmaeidae Patelloida latistrigata -87 ✖ ✔ Acmaeidae Patelloida cf. mufria Uncommon ✔ ✔ Haliotidae Haliotis ruber Rare (3) ✔ ✔ Rare Haliotidae Haliotis coccoradiata Rare (1) ✔ ✖ Rare Fissurellidae Scutus antipodes Common ✔ ✔ Rare Fissurellidae Amblychilepas nigrita Common ✔ ✖ Fissurellidae Diodora lineata Rare (2) ✖ ✖ Fissurellidae Montfortula rugosa Abundant ✔ ✔ Common Trochidae Austrocohlea constricta Abundant ✔ ✔ Common Trochidae Cantharidella picturata Abundant ✔ ✖ Rare Trochidae Tallorbis roseolus - ✖ 88 ✖ Rare Trochidae Clanculus brunneus - ✖ 3 ✖ Rare (1) Trochidae Clanculus floridus Rare (1) ✔ ✖ Trochidae Phasianotrochus eximius Uncommon ✔ ✖ Rare Trochidae Stomatella impertusa Uncommon ✔ ✖ Uncommon Trochidae Granata imbricata Uncommon ✖ 3 ✖ Cerithiidae Cacozeliana granaria Uncommon ✔ Uncommon Turbinidae Turbo undulatus Common ✔ ✔ Common Turbinidae Turbo torquatus Rare (2) ✖ 3 ✔ Uncommon Turbinidae Astralium tentoriformis Common ✔ ✔89 Uncommon Turbinidae Astralium rhodostomum Rare (1) ✔ ✖

86 Minchinton, T. E. 1996. Rocky intertidal shores. In Ecological study of Wollongong Harbour, North Wollongong Beach, Fairy Creek and Lagoon, and Stuart and J. P. Galvin Parks. Minchinton, T.E. and Whelan, R.J. Australian Flora and Fauna Research Centre, University of Wollongong. This study presents one species list for the Wollongong intertidal area. I have split the area into three study sites (Flagstaff Hill, North Wollongong Reef and Wollongong Channel). It is possible that some of the species listed by Minchinton and Whelan (1996) were not recorded on the Flagstaff Hill rock platform and reef. 87 I haven’t recorded this species at any site but the Wollongong region is within its’ range so it is likely to occur at several sites. 88 No live adults were found but the shells were. 89 Called Australium tentoriforme by Minchinton and Whelan (1996) Chapter 2 Appendix 2.1 421

Appendix 2.1i: North Wollongong continued. Family Species Occurrence Inventory Previous Eggs Shells studies90 Rissoidae Rissoina sp. Uncommon ✔ ✖ Rissoidae Rissoina (Rissolina) sp. Common ✔ ✖ Littorinidae Littorina unifasciata Abundant ✔ ✔ Littorinidae Littorina acutispira Abundant ✔ ✔ Littorinidae Nodilittorina pyramidalis Common ✔ ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ ✔ Common Cypraeidae Cypraea sp. Rare (1)91 ✖ ✖ Ranellidae Charonia lampas - ✖ ✔ Ranellidae Cabestana spengleri Common ✔ ✔ Uncommon Ranellidae Ranella australasia Rare (1) ✔ ✖ Rare Ranellidae Sassia parkinsonia Rare (2) ✖ ✖ Rare Muricidae Morula marginalba Abundant ✔ ✔ ✔ Common Muricidae Dicathais orbita Common ✔ ✔ ✔ Common Muricidae Agnewia tritoniformis Rare (2) ✔ ✖ ✔ Uncommon Mitridae Mitra sp. - ✖ ✖ Rare Conidae Conus sp. - ✖ ✖ Rare (1) Columbellidae Mitrella cf. semiconvexa Common ✔ ✖ Epitoniidae Opalia australis - ✖ ✖ Rare (1) Marginellidae Volvarina mustelina Common ✔ ✖ Siphonariidae Siphonaria denticulata Abundant ✔ ✔ ✔ Uncommon Siphonariidae Siphonaria funiculata Common ✔ ✖ Common Siphonariidae Siphonaria virgulata - ✖ ✔92 Siphonariidae Unidentified sp. - ✖ ✔93 Aplysiidae Aplysia sydneyensis Uncommon ✖ ✖ ✔ Aplysiidae Aplysia juliana Uncommon ✖ ✖ ✔ Aplysiidae Dolabrifera dolabrifera Rare (3) ✔ ✖ ✔ Pleurobranchidae Berthellina citrina Rare (1) ✖ Dendrodorididae Dendrodoris cf. fumata Rare (2) ✖ ✖ Dorididae Rostanga bassia Rare (5) ✖ ✖ Chromodoridae Hypselodoris bennetti Rare (1) ✔ ✖ Glaucidae Austraeolis ornata Rare (1) ✔94 ✖ ✔ Aeolidiidae Aeolidiella foulisi Uncommon ✔8 ✖ ✔ Ischnochitonidae Ischnochiton australis Common ✔ ✖ Uncommon Ischnochitonidae Ischnochiton versicolour Common ✔ ✖ Ischnochitonidae Ischnochiton elongatus Common ✔ ✖ Chitonidae Onithochiton quercinus Uncommon ✔ ✔ Rare Chitonidae Chiton pelliserpentis Common ✔ ✔ Rare Chitonidae Rhyssoplax jugosus Uncommon ✔ ✖ Mopalidae Plaxiphora albida Uncommon ✔ ✔ Rare

90 Minchinton, T. E. 1996. Rocky intertidal shores. In Ecological study of Wollongong Harbour, North Wollongong Beach, Fairy Creek and Lagoon, and Stuart and J. P. Galvin Parks. Minchinton, T.E. and Whelan, R.J. Australian Flora and Fauna Research Centre, University of Wollongong. This study presents one species list for the Wollongong intertidal area. I have split the area into three study sites (Flagstaff Hill, North Wollongong Reef and Wollongong Channel). It is possible that some of the species listed by Minchinton and Whelan (1996) were not recorded on the Flagstaff Hill rock platform and reef. 91 One large juvenile (4cm long), dark reddish mantle and brown shell 92 I have not observed this species in the Wollongong region. The species recorded by Minchinton and Whelan (1996) is most likely S. funiculata. 93 Species not identified by Minchinton and Whelan (1996) but could refer to S. zelandica, which is the only other Siphonariid likely to be found in the Wollongong Region 94 The adults and eggs of these species were found Chapter 2 Appendix 2.1 422

Appendix 2.1i: North Wollongong continued. Family Species Occurrence Inventory Previous Eggs Shells studies95 Acanthochitonidae Crytoplax mystica Uncommon ✔ ✖ Mytilidae Xenostrobus pulex Common ✔ ✖ Mytilidae Brachidontes rostratus Rare (3) ✖ ✖ Rare Mytilidae Trichomya hirsuta - ✖ ✖ Uncommon Anomiidae Anomia trigonopis - ✖ ✖ Rare Montacutidae Kellia rotunda Uncommon ✔ ✖ Ostreidae Saccostrea glomerata Uncommon ✔ ✔96 Sepiidae Sepia sp. - ✖ ✖ Uncommon Octopodidae Octopus sp. Rare ✔ ✖ Total number of species 6397 5498 29 11 4099

95 Minchinton, T. E. 1996. Rocky intertidal shores. In Ecological study of Wollongong Harbour, North Wollongong Beach, Fairy Creek and Lagoon, and Stuart and J. P. Galvin Parks. Minchinton, T.E. and Whelan, R.J. Australian Flora and Fauna Research Centre, University of Wollongong. This study presents one species list for the Wollongong intertidal area. I have split the area into three study sites (Flagstaff Hill, North Wollongong Reef and Wollongong Channel). It is possible that some of the species listed by Minchinton and Whelan (1996) were not recorded on the Flagstaff Hill rock platform and reef. 96 This species was recorded by Minchinton and Whelan (1996) as S. commercialis but almost definitely refers to S. glomerata 97 Number of species that were:- Abundant = 13; Common = 16; Uncommon = 15; Rare = 19 98 Live specimens were recorded for 52 species and the shells of a further 4 species were found washed up 99 The shells but no live specimens were found for nine species. Chapter 2 Appendix 2.1 423

Appendix 2.1j: Scarborough Location: Northern end of Wombarra-Scarborough Beach Habitat: This is an exposed rock platform with a steep drop-off into the ocean. There are a few shallow crevices and one large rock pool that has a sandy bottom. The lower intertidal areas are limited (primarily vertical) and difficult to access. The introduced species Caulerpa filiformis is dominant in the sublittoral zone and along the beach. It has also infiltrated the northern end of the platform in crevices and is abundant on the walls of the large rock pool. There is presently no fresh water run-off onto this reef but it is threatened by the approved development of a storm water drainage tunnel. Construction of the tunnel began in May, 1998. It is necessary to cross the beach to access this reef, so the beach debris was sampled more regularly here than at other sites. Inventory: 27/3/98; Tide = 0.1 Other sampling dates: 12/6/96; 27/6/97; 2/8/97; 10/8/97; 23/8/97; 28/12/97; 6/3/98; 6/3/98; 22/5/98; 7/6/98 Total number of sampling events: 11 Family Species Occurrence Previous Inventory Eggs Shells studies100 Neritidae Nerita atramentosa Abundant ✔ ✔ ✔ Common Patellidae Patella peronii Abundant ✖ ✔ Common Patellidae Patella chapmani - ✖ ✖ Common Patellidae Cellana tramoserica Abundant ✔ ✔ Common Acmaeidae Notoacmea petterdi Abundant ✖ ✔ Uncommon Acmaeidae Patelloida alticostata Common ✖ ✔ Uncommon Unidentified limpet - ✔ ✖ Haliotidae Haliotis ruber - ✖ ✖ Rare Haliotidae Haliotis coccoradiata - ✖ ✖ Rare Fissurellidae Diodora lineata - ✖ ✖ Rare (1) Fissurellidae Tugali sp. - ✖ ✖ Rare (1) Fissurellidae Montfortula rugosa Abundant ✔ ✔ Common Trochidae Austrocochlea constricta Abundant ✔ ✔ Common Trochidae Cantharidella picturata Common ✖ ✔ Uncommon Trochidae Tallorbis roseolus - ✖ ✖ Rare Trochidae Clanculus clangus - ✖ ✖ Rare Trochidae Clanculus brunneus - ✖ ✖ Rare Trochidae Clanculus floridus - ✖ ✖ Rare (1) Trochidae Phasianotrochus eximius Uncommon ✖ ✔ Uncommon Trochidae Granata imbricata - ✖ ✖ Rare (2) Turbinidae Turbo undulatus Common ✔ ✔ Common Turbinidae Turbo militaris - ✔ ✖ Turbinidae Ninella sp.101 - ✔2 ✖ Turbinidae Astralium rhodostomum - ✖ ✖ Rare (1) Littorinidae Littorina acutispira Abundant ✖ ✔ Littorinidae Littorina unifasciata Abundant ✔ ✔ Littorinidae Nodilittorina pyramidalis Abundant ✔ ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ ✔ Common Ranellidae Cabestana spengleri Rare (2) ✖ ✖ ✔ Uncommon 102 Ranellidae Ranella australasia - ✖ ✖ Rare (1) Muricidae Morula marginalba Abundant ✔ ✔ ✔3 Common

100 Marine Pollution Research Pty Ltd (1992). Marine Ecology of Scarborough/Wombarra Beach. Report on the Possible impacts of a proposed stormwater tunnel draining over Wombarra Beach. Report prepared for Snowy Mountains Engineering Corporation. 101 Ninella is a subgenus including only one species Turbo torquatus. Presumably this is the species recorded by Carolan et al., 1992 102 Hatched egg mass washed up on beach Chapter 2 Appendix 2.1 424

Appendix 2.1j: Scarborough continued. Family Species Occurrence Previous Inventory Eggs Shells studies103 Muricidae Dicathais orbita Common ✔ ✔ ✔ 104 Muricidae Lepsiella reticularis - ✖ ✖ ✔3 Muricidae Chicoreus sp. - ✖ ✖ Rare (1) Mitridae Mitra sp. - ✖ ✖ Rare Conidae Conus anemone - ✖ ✖ Rare (1) Conidae Conus paperliferus - ✖ ✖ Uncommon Conidae Conus sp. (eggs)105 - ✖ ✖ ✔3 Columbellidae Mitrella cf. semiconvexa - ✖ ✖ Uncommon Columbellidae Parviterebra trilineata - ✖ ✖ Rare (3) Epitoniidae Opalia australis - ✖ ✖ Rare (1) Janthinidae Janthina janthina - ✖ ✖ Uncommon Janthinidae Janthina exigua - ✖ ✖ Rare (1) Marginellidae Austroginella sp. - ✖ ✖ Uncommon Turridae Marita compta - ✖ ✖ Rare (1) Unidentified Unidentified eggs sp. 2106 - ✖ ✖ ✔3 Unidentified Unidentified eggs sp. 5107 - ✖ ✖ ✔3 Unidentified Unidentified eggs sp. 6108 - ✖ ✖ ✔3 Unidentified Unidentified eggs sp. 7109 - ✖ ✖ ✔3 Unidentified Unidentified eggs sp. 8110 - ✖ ✖ ✔3 Siphonariidae Siphonaria denticulata Abundant ✔ ✔ ✔ Uncommon Siphonariidae Siphonaria funiculata Abundant ✔ ✔ Common Siphonariidae Siphonaria zelandica Uncommon111 ✖ ✖ ✔ Uncommon Ischnochitonidae Ischnochiton australis Uncommon ✔ ✔ Uncommon Ischnochitonidae Ischnochiton versicolour Rare (5) ✖ ✔ Chitonidae Onithochiton quercinus Rare (3) ✔ ✔ Uncommon Chitonidae Chiton pelliserpentis Common ✔ ✔ Uncommon Chitonidae Rhyssoplax jugosus Rare (1) ✖ ✖ Mopalidae Plaxiphora albida Common ✔ ✔ Uncommon

103 Marine Pollution Research Pty Ltd (1992). Marine Ecology of Scarborough/Wombarra Beach. Report on the Possible impacts of a proposed stormwater tunnel draining over Wombarra Beach. Report prepared for Snowy Mountains Engineering Corporation. 104 Fresh and hatched egg capsules washed up on beach. 105 Some egg capsules resembling Conus spp. were found washed up on the beach. There was some variation in form and could possibly be the capsules of more than one species. 106 Flattened circular capsules with large pinkish eggs 107 Small white compartmentalised capsules found washed up on the beach. The capsules were found individually and conjoined. 108 Leathery egg capsules with spikes (3 peaks on top). These eggs are could be a Muricid because some of the capsules were stained purple. 109 Purple stained egg capsules, which are frilly around the top and there is no escape aperture. 110 One sheet of conjoined egg capsules found washed up on the beach. The individual capsules are small (~ 1mm diameter) and have clear escape apertures at the top. Most capsules were hatched but some contained orange eggs. 111 The populations of these species fluctuate over time. They are not recorded on every field trip. Chapter 2 Appendix 2.1 425

Appendix 2.1j: Scarborough continued. Family Species Occurrence Previous Inventory Eggs Shells studies112 Mytilidae Xenostrobus pulex Common11 ✖ ✖ Mytilidae Trichomya hirsuta - ✖ ✖ Uncommon Anomiidae Anomia trigonopis - ✖ ✖ Uncommon Ostreidae Saccostrea glomerata Abundant ✔ ✔ Sepiidae Sepia sp. - ✖ ✖ Uncommon Octopodidae Octopus sp. Rare ✖ ✖ Total number of species 29113 20 24 14114 46115

112 Marine Pollution Research Pty Ltd (1992). Marine Ecology of Scarborough/Wombarra Beach. Report on the Possible impacts of a proposed stormwater tunnel draining over Wombarra Beach. Report prepared for Snowy Mountains Engineering Corporation. 113 Number of species that were:- Abundant = 14; Common = 7; Uncommon = 3; Rare = 5 One species that was recorded as common and another recorded as uncommon, have fluctuating populations and were not always recorded. 114 The egg masses of only four species were found on the intertidal rock platform. However, the egg capsules of a further 10 species were found washed up on the beach. 115 The shells of 25 species, which were not found live, were found washed up on the beach. The shells of two species were more common than the live specimens. Chapter 2 Appendix 2.1 426

Appendix 2.1k: South Shellharbour Location: On the northern side of Bass Point from the south eastern end of Shellharbour South Beach to the gravel loader. Habitat: This is a sheltered low relief boulder-field with rocky outcrops and many shallow, boulder filled hollows that retain water on even the lowest tides. It is the largest uninterrupted boulder field in the Wollongong Region. This reef is potentially threatened by indirect impacts from the approved development of a 350-berth boatharbour/marina. Inventory: 29/1/98; Tide = 0.1 Other sampling dates: 29/9/95; 5/10/95; 23/10/95; 17/10/96; 12/11/96; 23/1/97; 6/3/97; 22/4/97; 5/7/97; 19/8/97; 14/9/97; 14/2/98; 9/4/98; 8/5/98 Total number of sampling events: 15

Family Species Occurrence Inventory Previous Eggs Shells studies116 Architectonicidae Philippia lutea Rare (3) ✔ ! Neritidae Nerita atramentosa Abundant ✔ ✔ ✔ Common Patellidae Patella peronii Abundant ✔ ✔ Common Patellidae Patella chapmani - ! ! Common Patellidae Cellana tramoserica Abundant ✔ ✔ Common Acmaeidae Notoacmea petterdi Abundant ✔ ! Uncommon Acmaeidae Patelloida alticostata Common ✔ ! Common Acmaeidae Patelloida cf. mufria Common ✔ ! Haliotidae Haliotis ruber Uncommon ! ! Uncommon Haliotidae Haliotis coccoradiata Uncommon ✔ ! Rare Haliotidae Haliotis cf. melculus - ! ! Rare (2) Fissurellidae Scutus antipodes Common ✔ ! Rare Fissurellidae Amblychilepas nigrita Uncommon ✔ ! Fissurellidae Diodora lineata Common ✔ ! Fissurellidae Montfortula rugosa Abundant ✔ ! Common Trochidae Austrocochlea constricta Abundant ✔ ✔ Common Trochidae Cantharidella picturata Abundant ✔ ! Uncommon Trochidae Clanculus clangus Rare (3) ✔ ! Rare Trochidae Clanculus brunneus Common ✔ ! Rare Trochidae Clanculus floridus Rare (5) ! ! Rare Trochidae Granata imbricata Common ✔ ! Uncommon Trochidae Herpetopoma aspersa Uncommon ✔ ! Trochidae Phasianotrochus eximius Common ✔ ! Uncommon Trochidae Phasianotrochus sp.117 Rare (1) ✔ ! Rare (1) Trochidae Stomatella impertusa Common ✔ ! Trochidae Tallorbis roseolus Common ✔ ! Rare Turbinidae Astralium rhodostomum Rare ✔ ! Turbinidae Astralium squamiferum Uncommon ✔ ! Rare Turbinidae Astralium tentoriformis Abundant ✔ ✔ Common Turbinidae Turbo undulatus Abundant ✔ ✔ Common Turbinidae Turbo torquatus Uncommon ✔ ! Uncommon Turbinidae Phasianella sp. Rare ✔ ! Cerithiidae Cacozeliana granaria Abundant ✔ ! Common Dialidae Diala sp. Rare (2) ! !

116 Marine Pollution Research Pty Ltd. 1995. Ecological survey and assessment of marine ecological impacts. Appendix 11 Environmental Impact Statement: Shell Cove Boatharbour/Marina. Lester Firth (aust) Pty Ltd. Edgecliff, NSW, Australia. This site was called Shellharbour North by Marine Pollution Research, 1995. 117 Small (6mm) yellow/ orange shell Chapter 2 Appendix 2.1 427

Appendix 2.1k: South Shellharbour Family Species Occurrence Inventory Previous Eggs Shells studies118 Planaxidae Hinea brasiliana Uncommon ! ! Littorinidae Bembicium nanum Abundant ✔ ✔ ✔ Common Littorinidae Littorina acutispira Abundant ✔ ! Littorinidae Littorina unifasciata Abundant ✔ ✔ Littorinidae Nodilittorina pyramidalis Abundant ✔ ! Rissoidae Rissoina sp. Abundant ✔ ! Common Rissoidae Rissoina (Rissolina) sp. Common ✔ ! Common Vanikoridae Vanikora sigaretiformis Rare (1) ✔ ! Cypraeidae Cypraea caputserpentis Uncommon !119 ! Uncommon Cypraeidae Cypraea clandestina Rare (1) ✔ ! Rare (1) Cypraeidae Cypraea flaveola Rare (1) ! ! Cypraeidae Cypraea vitellus - ! ! Rare (1) Cypraeidae Cypraea sp.120 Rare (1) ! ! Cypraeidae Cypraea juveniles sp. 1121 Rare (3) ! ! Cypraeidae Cypraea juveniles sp. 2 122 Rare (3) ! ! Triviinae Trivia merces Rare (1) ! ! Uncommon Cassidae Semicassis labiatum Rare (5)123 ! ! Uncommon124 Ranellidae Cabestana spenglerii Common ✔ ✔ ✔125 Common Ranellidae Charonia lampas Uncommon ✔ ! Ranellidae Cymatium parthenopeum Rare (1) ✔ ! Ranellidae Ranella australasia Uncommon ✔ ! Uncommon Ranellidae Sassia parkinsonia Common ✔ ! Uncommon Epitoniidae Epitonium sp. Uncommon ✔ ! Uncommon Epitoniidae Opalia australis Rare (2) ! ! Rare Epitoniidae Opalia ballinensins Rare (1) ✔ ! Rare Janthinidae Janthina janthina - ! ! Uncommon Muricidae Agnewia tritoniformis Common ✔ ! ✔ Uncommon Muricidae Bedeva hanleyi Uncommon ✔126 ! ✔ Rare Muricidae Dicathais orbita Common ✔ ! ✔5 Common Muricidae Ergalatax contracta Uncommon ✔6 ! ✔ Muricidae Lepsiella reticulata Common ✔ ! Rare Muricidae Morula marginalba Abundant ✔ ✔ ✔ Common Muricidae Phyllocoma speciosa Rare (1) ! ! Buccinidae Cominella eburnea Rare (3) ✔ ! Buccinidae Engina australis Common ✔ ! Rare Buccinidae Fractolatirus normalis - ! ! Rare Buccinidae Nassarius jonasii Uncommon ✔ ! Mitridae Mitra boudi Uncommon ✔ ! ✔ Rare Mitridae Mitra carbonaria Uncommon ! ! ✔ Rare Mitridae Mitra glabra - ! ! Rare Conidae Conus anemone Rare (4) ! ! Uncommon Conidae Conus paperliferus Uncommon ✔6 ! ✔ Columbellidae Euplica versicolour Rare (2) ! ! Columbellidae Mitrella cf. semiconvexa Common ✔ !

118 Marine Pollution Research Pty Ltd. 1995. Ecological survey and assessment of marine ecological impacts. Appendix 11 Environmental Impact Statement: Shell Cove Boatharbour/Marina. Lester Firth (aust) Pty Ltd. Edgecliff, NSW, Australia. This site was called Shellharbour North by Marine Pollution Research, 1995. 119 No live specimens but one shell found. 120 large white shell, orange flesh 121 brown shell to 3-6cm 122 white shell 1-2cm, some with yellow stripe 123 Two adults and three juveniles 124 Many adult shells and two juvenile shells 125 Hatched egg capsules found washed up. 126 Adults and eggs found Chapter 2 Appendix 2.1 428

Appendix 2.1k: South Shellharbour Family Species Occurrence Inventory Previous Eggs Shells studies127 Columbellidae Parviterebra sp. Rare (2) ! ! Marginellidae Austroginella sp. Uncommon ✔ ! Uncommon Marginellidae Volvarina mustelina Common ✔ ! Turridae Marita compta - ! ! Rare Unidentified Unidentified eggs sp. 2128 Uncommon ! ! ✔ Siphonariidae Siphonaria denticulata Abundant ✔ ! ✔ Uncommon Siphonariidae Siphonaria funiculata Abundant ✔ ! Common Siphonariidae Siphonaria zelandica Common ✔ ! ✔ Onchidiidae Onchidella patelloides Rare (2) ! ! Aplysiidae Aplysia dactylomela Uncommon ! ✔ ✔ Aplysiidae Aplysia juliana Rare (2) ! ! Aplysiidae Aplysia parvula Uncommon ✔ ! ✔ Aplysiidae Aplysia sydneyensis Uncommon ! ! ✔ Aplysiidae Dolabrifera dolabrifera Common ✔6 ! ✔ Aplysiidae Stylocheilus longicauda129 Uncommon ! ! ✔ Pleurobranchidae Berthellina citrina Uncommon ✔ ! ✔ Rare Pleurbranchidae Pleurobranchus peroni Rare ! ! Pleurobranchidae Pleurobranchea sp. Uncommon ! ! ✔ Bullinidae Bullina lineata Uncommon ✔ ! ✔ Rare Oxynoidae Oxynoe viridis Rare ! ! Elysiidae Elysia australis Uncommon ! ! ✔ Dendrodorididae Dendrodoris fumata Uncommon ! ! ✔ Dendrodorididae Dendrodoris nigra Uncommon ✔6 ! ✔ Dendrodorididae Doriopsilla miniata Uncommon ✔6 ! ✔ Dendrodorididae Doriopsilla carneola Rare (2) ! ! Dorididae Discodoris fragilis Uncommon ✔6 ! ✔ Dorididae Platyodoris sp. Uncommon ✔ ! ✔ Dorididae Hoplodoris nodulosa Rare (2) ✔ ! Dorididae Jorunna pantherina Common ✔ ! ✔ Dorididae Rostanga bassia Common ✔ ! ✔ Chromodorididae Ceratosoma amoena Rare (1) ✔ ! Chromodorididae Hypselodoris bennetti Rare (2) ✔ ! Polyceridae Plocampherus imperialis Rare (2) ✔ ! Goniodorididae Goniodoris sp. Uncommon ! ! ✔ Glaucidae Austraeolis ornata Uncommon ✔6 ! Glaucidae Glaucus atlanticus Uncommon ! ! Glaucidae Glaucilla marginata Rare (3) ! ! Aeolidiidae Aeolidiella foulisi Common ✔6 ! Caliphyllidae Cyerce sp. 130 Rare (1) ! ! Unidentified Unidentified sp. 1131 Rare (3) ! ! ✔ Unidentified Unidentified eggs sp. 10132 Uncommon ! ! ✔ Ischnochitonidae Callochiton crocina Uncommon ✔ ! Ischnochitonidae Ischnochiton australis Abundant ✔ ! Ischnochitonidae Ischnochiton elongatus Common ✔ ! Ischnochitonidae Ischnochiton versicolour Abundant ✔ ! Chitonidae Chiton pelliserpentis Common ✔ !

127 Marine Pollution Research Pty Ltd. 1995. Ecological survey and assessment of marine ecological impacts. Appendix 11 Environmental Impact Statement: Shell Cove Boatharbour/Marina. Lester Firth (aust) Pty Ltd. Edgecliff, NSW, Australia. This site was called Shellharbour North by Marine Pollution Research, 1995. 128 Flattened circular capsules with large pinkish eggs 129 Could also be Bursatella leachii. I find these two species difficult to distinguish and they were larger than the Stylocheilus I find in Wollongong Channel. 130 Orange body covered with black triangular projections 131 Small white nudibranch with green gills 132 White spiralled ribbon flattened onto rock surface Chapter 2 Appendix 2.1 429

Appendix 2.1k: South Shellharbour Family Species Occurrence Inventory Previous Eggs Shells studies133 Chitonidae Onithochiton quercinus Abundant ✔ ! Chitonidae Rhyssoplax jugosus Common ✔ ! Mopalidae Plaxiphora albida Uncommon ! ! Acanthochitonidae Crytoplax mystica Common ✔ ! Acanthochitonidae Unidentified sp. Rare (1) ✔ ! Mytilidae Mytilus edulis Uncommon ✔ ! Rare Mytilidae Trichomya hirsuta Common ✔ ! Common Pectinidae Scaeochlamys livida Common ✔ ! Anomiidae Anomia trigonopis Uncommon ! ! Uncommon Limidae Limatula strangei Rare (4) ! ! Rare Montacutidae Kellia rotunda Common ✔ ! Ostreidae Saccostrea glomerata Abundant ✔ ! Ostreidae Unidentified sp.134 Rare (1) ! ! Sepiidae Sepia sp. - ! ! Uncommon Octopodidae Hapalochlaena maculosa Rare (3) ✔ ! Octopodidae Octopus sp. Uncommon ✔ ! Total Number of Species 131135 94 11 34136 96137

133 Marine Pollution Research Pty Ltd. 1995. Ecological survey and assessment of marine ecological impacts. Appendix 11 Environmental Impact Statement: Shell Cove Boatharbour/Marina. Lester Firth (aust) Pty Ltd. Edgecliff, NSW, Australia. This site was called Shellharbour North by Marine Pollution Research, 1995. 134 Fragile white shell with red and blue markings 135 Number of species that were: Abundant 22; Common 30; Uncommon 41; Rare 38 (incl. 2 unidentified egg masses and 2 unidentified juveniles). 136 The egg masses from 30 species were found in the intertidal area. The hatched egg capsules of a further two species were found washed up. 137 No live specimens but the shells of nine species were found. Chapter 2 Appendix 2.1 430

Appendix 2.1l: Towradgi Location: Towradgi point, around Towradgi swimming pool at the northern end of Towradgi-Fairy Meadow Beach. Habitat: This is a large wave exposed boulder field with several rocky outcrops and a small rock platform. Most of the boulder field is dry during low tide but there are some water retaining hollows. Many of the boulders are fixed into position by the growth of encrusting alga. There are some sheltered sandy patches in between the natural rock platform and the artificial swimming pool (refer to Appendix 2.2f). Much of the lower intertidal and sublittoral zone is dominated by the growth of the introduced algae Caulerpa filiformis. Inventory: 28/1/98; Tide = 0.2 Other sampling dates: 14/8/95; 15/8/95; 9/9/95; 18/8/96; 23/8/96; 12/9/96; 26/9/96; 3/10/96; 24/10/95; 25/10/96; 28/5/97; 1/7/97; 4/8/97; 20/8/97; 15/9/97; 17/10/97; 13/12/97; 27/2/98; 25/4/98. Total number of sampling events: 20 Family Species Occurrence Inventory Eggs Shells Neritidae Nerita atramentosa Abundant ✔ ✔ Common Patellidae Patella peronii Abundant ✔ Common Patellidae Patella chapmani - ✖ Uncommon Patellidae Cellana tramoserica Abundant ✔ Common Acmaeidae Notoacmea petterdi Uncommon ✔ Acmaeidae Patelloida alticostata Common ✔ Uncommon Acmaeidae Patelloida cf. mufria Rare (5) ✖ Haliotidae Haliotis ruber Rare (1) ✖ Haliotidae Haliotis coccoradiata - ✖ 138 Rare (1) Fissurellidae Scutus antipodes Uncommon ✔ Uncommon Fissurellidae Amblychilepas nigrita Rare (3) ✖ Fissurellidae Montfortula rugosa Abundant ✔ Trochidae Austrocochlea constricta Abundant ✔ Common Trochidae Cantharidella picturata Abundant ✔ ✔ Uncommon Trochoidea Tallorbis roseolus - ✖ Rare Trochidae Clanculus sp. - ✖ Rare Trochidae Phasianotrochus eximius Uncommon ✔ Rare Trochidae Granata imbricata - ✖ 1 Rare Turbinidae Turbo undulatus Abundant ✔ Common Batillariidae Velacumantus australis - ✖ Rare Cerithiidae Unidentified egg mass139 Rare (1) ✖ ✔ Littorinidae Littorina acutispira Abundant ✔ Littorinidae Littorina unifasciata Abundant ✔ Littorinidae Nodilittorina pyramidalis Uncommon ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ Common Rissoidae Rissoina (Rissolina) sp. Uncommon ✔ Uncommon Cypraeidae Cypraea cf. clandestina - ✖ Rare (1) Ranellidae Cabestana spengleri Common ✔ ✔ Common Ranellidae Ranella australasia - ✖ Rare Ranellidae Cymatium parthenopeum Rare (1) ✔ Janthinidae Janthina janthina - ✖ Uncommon Janthinidae Janthina exigua - ✖ Rare (1) Muricidae Morula marginalba Abundant ✔ Common

138 No live specimens but one shell was found 139 Long (>20cm), thin (<1cm) white tubular capsules with large eggs thinly dispersed, most likely deposited by a large Cerithiidae (Ian Loch, pers. comm.) Chapter 2 Appendix 2.2 431

Appendix 2.1l: Towradgi continued. Family Species Occurrence Inventory Eggs Shells Muricidae Dicathais orbita Abundant ✔ ✔ Common Muricidae Lepsiella reticulata Uncommon ✔140 ✔ Rare Conidae Conus paperliferus Rare (1) ✔ Conidae Conus sp. egg capsules - ✖ ✔141 Columbellidae Mitrella cf. semiconvexa Common ✔ Unidentified Unidentified eggs sp. 6142 - ✖ ✔143 Siphonariidae Siphonaria denticulata Abundant ✔ ✔ Common Siphonariidae Siphonaria funiculata Common ✔ Common Aplysiidae Aplysia juliana Common ✔ ✔ Pleurobranchidae Pleurobranchea sp. Rare (3) ✖ ✔ Bullinidae Bullina lineata (eggs only) Rare (2) ✔ ✔ Hydatinidae Hydatina physis Rare (1) ✖ Rare (1) Umbraculidae Umbraculum sp. Rare (1) ✖ Oxynoidae Oxynoe viridis Rare (1) ✖ ✔144 Elysiidae Elysia australis (eggs only) Uncommon ✔ ✔ Dendrodorididae Dendrodoris sp. Rare (1) ✔ Dendrodorididae Doriopsilla carneola Rare (1) ✖ Dendrodorididae Doriopsilla miniata Rare (1) ✖ Dorididae Hoplodoris nodulosa Rare (2) ✖ Dorididae Platyodoris sp. Rare (1) ✔ Dorididae Rostanga bassia Common ✔ ✔ Dorididae Thordisa sp. Rare (2) ✖ Chromodoridae Hypselodoris bennetti Uncommon ✔ ✔145 Polyceridae Plocamopherus imperialis Rare (2) ✖ Polyceridae Polycerid sp.146 Rare (1) ✖ Goniodoridae Goniodoris sp. Common ✖ ✔ Glaucidae Austraeolis ornata Rare ✖ Glaucidae Spurilla sp. Rare (1) ✖ Aeolidiidae Aeolidiella foulisi Common ✔3 ✔ Ischnochitonidae Ischnochiton australis Abundant ✔ Ischnochitonidae Ischnochiton versicolour Abundant ✔ Ischnochitonidae Ischnochiton elongatus Abundant ✔ Chitonidae Onithochiton quercinus Rare (2) ✖ Chitonidae Chiton pelliserpentis Uncommon ✔ Chitonidae Rhyssoplax jugosus Uncommon ✔ Mopalidae Plaxiphora albida Common ✔ Uncommon

140 Adults and eggs found 141 A couple of egg capsules resembling Conus spp. were found washed up on the beach. 142 Conjoined spiky leathery egg capsules, stained purple. 143 Only found among beach debris. 144 I found a white spiralled egg ribbon attached to Caulerpa filiformis with an adult but I can not be certain it belongs to this species 145 I have seen egg ribbons that match previous descriptions of egg ribbons from this species but I was unable to confirm the identity because this species does not lay in aquaria 146 Unnamed species (Bill Rudman, personal communication).

Chapter 2 Appendix 2.2 432

Appendix 2.1l: Towradgi continued. Family Species Occurrence Inventory Eggs Shells Mytilidae Trichomya hirsuta Uncommon ✖ 147 Uncommon Anomiidae Anomia trigonopis - ✖ 10 Uncommon Montacutidae Kellia rotunda Uncommon ✔ Ostreidae Saccostrea glomerata Uncommon ✔ Sepiidae Sepia sp. - ✖ Uncommon Octopodidae Octopus sp. Rare ✔ Total number of species 60148 42149 19150 32151

147 No live adults but several shells were found 148 Number of species that were:- Abundant = 16; Common = 9; Uncommon = 13; Rare = 22 One uncommon species and two rare species were only recorded from the presence of egg masses. 149 Forty two species were recorded from the presence of live specimens but two of these were egg masses. A further four species were recorded from the presence of shells. 150 The egg masses from 17 species were found in the intertidal area. The hatched egg capsules from a further two species were found washed up o the beach. 151 The shells of 12 species were found, which were not recorded live.

Chapter 2 Appendix 2.2 433

Appendix 2.1m: Wombarra Location: On the southern end of Wombarra Beach, around Wombarra swimming pool. Habitat: This is a large wave exposed rock platform with steep drop-offs into the ocean. There is an off shore reef with many channels and natural rock pools containing boulders, to the east of the rock platform. This reef is only accessible on very calm low tides and was only sampled three times during the study period. Nevertheless, a number of species were only recorded on the off-shore reef. There is an artificial swimming pool on the rock platform (refer to Appendix 2.2h) and to the north of this, the reef slopes fairly gently into the ocean. There are a few boulders in shallow water retaining depressions in this area but they shift around a lot in the swell. This whole area becomes periodically inundated with sand and is heavily impacted by fresh water run-off. There are two storm water drains and a creek that spill onto Wombarra reef. The introduced species Caulerpa filiformis is dominant in the sublittoral zone, particularly on the northern edge of the rock platform and in between the platform and the off-shore reef. Some C. filiformis has also infiltrated the crevices on the rock platform. Inventory: 30/12/97; Tide = 0.2 Other sampling dates: 31/11/96; 28/5/97; 13/6/97; 7/7/97; 13/9/97; 25/10/97; 1/11/97; 8/11/97; 23/11/97; 9/1/98; 7/3/98; 17/4/98; 22/5/98; 21/6/98 Total number of sampling events: 15 Family Species Occurrence Inventory Previous Eggs Shells studies152 Architectonicidae Philippia lutea Rare (5)153 ✔ ! Neritidae Nerita atramentosa Abundant ✔ ✔ ✔ Common Patellidae Patella peronii Abundant ✔ ! Common Patellidae Patella chapmani Uncommon ✔ ! Common Patellidae Cellana tramoserica Abundant ✔ ✔ Common Unidentified limpet - ! ✔ Acmaeidae Notoacmea petterdi Abundant ✔ ! Uncommon Acmaeidae Patelloida alticostata Common ✔ ! Uncommon Haliotidae Haliotis ruber Uncommon2 ✔ ! Haliotidae Haliotis coccoradiata Rare (1)2 ! ! Rare (2) Fissurellidae Scutus antipodes Common ✔ ! Uncommon Fissurellidae Amblychilepas nigrita Uncommon ✔ ! Fissurellidae Diodora lineata Uncommon2 ! ! Rare Fissurellidae Tugali sp. - ! ! Rare (1) Fissurellidae Montfortula rugosa Abundant ✔ ✔ Common Trochidae Austrocohlea constricta Abundant ✔ ✔ Common Trochidae Cantharidella picturata Abundant ✔ ! Uncommon Trochidae Tallorbis roseolus Uncommon2 ✔ ! Rare Trochidae Clanculus clangus - ! ! Rare Trochidae Clanculus brunneus - ! ! Rare Trochidae Clanculus floridus Rare (1) 2 ! ! Trochidae Phasianotrochus eximius Common ✔ ! Rare Trochidae Granata imbricata Rare (1) 2 ! ! Rare Turbinidae Turbo undulatus Common ✔ ✔ Common Turbinidae Turbo torquatus Rare2 ! ✔ Turbinidae Astralium tentoriformis Uncommon2 ✔ ! Rare

152 Marine Pollution Research Pty Ltd (1992). Marine Ecology of Scarborough/Wombarra Beach. Report on the Possible impacts of a proposed stormwater tunnel draining over Wombarra Beach. Report prepared for Snowy Mountains Engineering Corporation. 153 Only recorded on the off-shore reef.

Chapter 2 Appendix 2.2 434

Appendix 2.1m: Wombarra continued. Family Species Occurrence Inventory Previous Eggs Shells studies154 Turbinidae Astralium squamiferum - ! ! Rare (1) Turbinidae Ninella sp.155 - ! ✔ Littorinidae Littorina acutispira Abundant ✔ ! Littorinidae Littorina unifasciata Abundant ✔ ✔ Littorinidae Nodilittorina pyramidalis Abundant ✔ ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ ✔ Common Cassidae Semicassis labiatum - ! ! Rare (1) Cymatiidae Cabestana spengleri Common ✔ ! ✔ Uncommon 156 Cymatiidae Ranella australasia Rare (1) 2 ✔ ! Rare Muricidae Morula marginalba Abundant ✔ ✔ Common Muricidae Dicathais orbita Common ✔ ✔ ✔5 Common Muricidae Lepsiella reticularis - ! ! ✔5 Mitridae Mitra sp. - ! ! Rare Conidae Conus sp. - ! ! ✔5 Rare Columbellidae Mitrella sp. Common ✔ ! Epitoniidae Opalia australis - ! ! Rare Janthinidae Janthina janthina - ! ! Uncommon Unidentified Unidentified eggs sp. 5157 - ! ! ✔5 Unidentified Unidentified eggs sp. 7158 - ! ! ✔5 Siphonariidae Siphonaria denticulata Abundant ✔ ✔ ✔ Uncommon Siphonariidae Siphonaria funiculata Common ✔ ✔ Uncommon Siphonariidae Siphonaria zelandica Abundant ✔ ! ✔ Aplysiidae Aplysia juliana Common ! ! ✔ Aplysiidae Aplysia parvula Uncommon ! ! ✔ Pleurobranchidae Pleurobranchea sp. Rare (1) ! ! Goniodoridae Goniodoris sp. Uncommon ! ! Glaucidae Austraeolis ornata Rare (1) ! ! Ischnochitonidae Ischnochiton australis Common ✔ ! Ischnochitonidae Ischnochiton versicolour Uncommon ✔ ! Ischnochitonidae Ischnochiton elongatus Uncommon ✔ ! Chitonidae Onithochiton quercinus Uncommon ! ✔ Chitonidae Chiton pelliserpentis Common ✔ ! Mopalidae Plaxiphora albida Uncommon ✔ ! Mytilidae Xenostrobus pulex Common ✔ ! Mytilidae Trichomya hirsuta Rare2 ! ! Uncommon Anomiidae Anomia trigonopis - ! ! Rare Ostreidae Saccostrea glomerata Common ✔ ✔ Sepiidae Sepia sp. - ! ! Octopodidae Octopus sp. Rare (2) 2 ! ! Total number of species 49159 36 17 12 36161 160

154 Marine Pollution Research Pty Ltd (1992). Marine Ecology of Scarborough/Wombarra Beach. Report on the Possible impacts of a proposed stormwater tunnel draining over Wombarra Beach. Report prepared for Snowy Mountains Engineering Corporation. 155 Ninella is a subgenus including only one species Turbo torquatus. Presumably this is the species recorded by MPR, 1992. 156 Hatched egg mass washed up on beach. 157 Small white compartmentalised capsules found washed up on the beach. 158 Some purple stained leathery egg capsules were found washed up on the beach. These capsules are frilly around the top and there is no escape aperture. 159 The number of species that were:- Abundant = 14; Common = 13; Uncommon = 12; Rare = 10 160 The egg masses from six species were found in the intertidal area and the hatched egg capsules from a further six species were found washed up on the shore. 161 The shells of 13 species, which were not found live, were found among beach debris.

Chapter 2 Appendix 2.2 435

Appendix 2.2

Species List of Molluscs from nine artificial habitats on the Wollongong Coast, NSW, Australia.

The species of molluscs recorded from 8 artificial swimming pools and one artificially sheltered channel along the Wollongong Coast, NSW, Australia. The species are listed for both a single inventory and the accumulated records from 1995-1998. A description of the habitat and the sampling dates are provided for each site. Records of molluscan egg masses are also included.

The occurrence of each species refers to its estimated abundance at that site (refer to Methods, Section 2.3.2).

Appendix 2.2a: Austinmer Pool Appendix 2.2b: Bellambi Pool Appendix 2.2c: Bulli Pool Appendix 2.2d: Coalcliff Pool Appendix 2.2e: Coledale Pool Appendix 2.2f: Towradgi Pool Appendix 2.2g: Wollongong Pools Appendix 2.2h: Wombarra Pool Appendix 2.2i: Wollongong Channel

Chapter 2 Appendix 2.2 436

Appendix 2.2a: Austinmer Pools Habitat: On the south-end of Austinmer Beach is an exposed rock platform with two large man-made pools that are not painted. The northern pool is regularly cleaned. There is also a shallow pool, which has been cut out of the rock platform. All threes pools have sandy bottoms, although the level of sand fluctuates. Inventory: 14/12/97, Tide = 0.2 Other sampling dates: 4/4/95; 10/4/95; 27/4/95; 15/5/95; 19/5/95; 20/5/95 26/7/95; 19/9/95; 26/7/96; 31/11/96; 7/6/97; 8/6/97; 5/7/97; 16/11/97; 8/1/98; 20/2/98 (night); 3/4/98; 8/5/98, 13/6/98. Total number of sampling periods: 20

Family Species Occurrence Inventory Eggs Neritidae Nerita atramentosa Common ✔ ✔ Patellidae Patella peronii Uncommon ✔ Patellidae Cellana tramoserica Common ✔ Fissurellidae Montfortula rugosa Common ✔ Trochidae Austrocohlea constricta Uncommon ✔ Trochidae Cantharidella picturata Uncommon1 ✖ Trochidae Phasianotrochus eximius Rare1 ✖ Turbinidae Turbo undulatus Uncommon162 ✖ Littorinidae Littorina unifasciata Abundant ✔ Littorinidae Bembicium nanum Common ✔ ✔ Muricidae Morula marginalba Common1 ✔ Muricidae Dicathais orbita Common1,163 ✖ Siphonariidae Siphonaria denticulata Uncommon ✔ Siphonariidae Siphonaria funiculata Common ✔ Aplysiidae Aplysia sydneyensis Common164 ✖ ✔ Aplysiidae Aplysia juliana Common3 ✖ ✔ Aplysiidae Aplysia parvula Rare ✖ Bullinidae Bullina lineata Rare ✖ ✔ (3) (eggs only)165 Hydatinidae Hydatina physis Rare (1) ✖ ✔ (1) (eggs only)4 Ostreidae Saccostrea glomerata Common ✔ Total number of species 20166 11 6

162 Only in large pool that is not regularly cleaned 163 Mostly juveniles 164 These species have fluctuating populations and were not always recorded 165 Only in shallow sandy pool on the southern rock platform 166 A different number of species were recorded for the different pools. Large pool regularly cleaned = 13 species Large pool not regularly cleaned = 18 species Shallow pool = 20 species In total, the number of species that were:- Abundant = 1; Common = 10; Uncommon = 5; Rare = 4 Two of the common species were not recorded on every occasion. Two of the rare species were only recorded from the presence of their egg masses

Chapter 2 Appendix 2.2 437

Appendix 2.2b: Bellambi Pool Habitat: Large artificial swimming pool painted with antifouling paint on the northern end of Bellambi reef. The pool is regularly cleaned. The bottom of the pool is cement but it does collect some sand. Inventory: 1/2/98, Tide =0.2 Other sampling dates: 22/10/96; 30/10/96; 2/11/96; 9/12/96; 10/12/96; 23/4/97; 24/4/97; 4/7/97; 19/7/97; 3/8/97; 18/8/97; 19/10/97; 29/11/97; 31/12/97 Total number of sampling periods: 15

Family Species Occurrence Inventory Eggs Neritidae Nerita atramentosa Common ✔ ✔ Patellidae Patella peronii Uncommon1 ! Patellidae Cellana tramoserica Uncommon ✔ 167 Fissurellidae Montfortula rugosa Common1 ✔ Trochidae Austrocohlea constricta Common ✔ Littorinidae Nodilittorina unifasciata Abundant ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ Muricidae Morula marginalba Common168 ✔ Siphonariidae Siphonaria denticulata Uncommon ✔ ✔ 169 Siphonariidae Siphonaria funiculata Common3 ✔ Aplysiidae Aplysia sydneyensis Common170 ! ✔ Aplysiidae Aplysia juliana Abundant4 ✔ ✔ Aplysiidae Stylocheilus longicauda Uncommon4 ! ✔ Philinidae Philine angasi Uncommon ! ✔ 171 Total number of species 14172 10 7

167 Uncommon in pool but more common on the back wall 168 Only on the outside of the eastern wall ie. facing the ocean 169 Only on the eastern wall (both sides but more common on sea-side) 170 These species have fluctuating populations and were not recorded on every trip 171 This species was only recorded on one occasion, when the pool was drained. They were found on the bottom of the pool in sand. 172 Number of species that were:- Abundant = 3; Common = 6; Uncommon = 5; Rare = 0

Chapter 2 Appendix 2.2 438

Appendix 2.2c: Bulli Pool Habitat: One large artificial swimming pool and one shallow small pool. The large pool is not painted with antifouling paint but the small pool is. Both are cleaned regularly. Both pools have a cement bottom but this is usually covered with sand. Inventory: 27/1/98; Tide = 0.2 Other sampling dates: 10/4/95; 18/10/95; 17/8/96; 10/10/96; 4/11/96; 19/11/96; 27/11/96; 28/11/96; 5/4/97; 28/5/96; 4/7/97; 1/8/97; 19/9/97; 27/10/97; 2/11/97; 16/12/97, 31/1/98, 26/4/98, 13/6/98 Total number of sampling events: 20

Family Species Occurrence Inventory Eggs Neritidae Nerita atramentosa Abundant ✔ ✔ Patellidae Patella peronii Common173 ✔ Patellidae Cellana tramoserica Common1 ✔ Fissurellidae Montfortula rugosa Common1 ✔ Trochidae Austrocohlea constricta Common ✔ Littorinidae Littorina unifasciata Abundant ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ Nassaridae Nassarius jonasii Common174 ✖ Muricidae Morula marginalba Common1 ✔ Muricidae Dicathais orbita Common1,175 ✖ Siphonariidae Siphonaria denticulata Uncommon ✔ ✔ Siphonariidae Siphonaria funiculata Common176 ✔ Aplysiidae Aplysia sydneyensis Common177 ✖ ✔ Aplysiidae Aplysia juliana Abundant5 ✖ ✔ Aplysiidae Stylocheilus longicauda Common5 ✖ ✔ Aplysiidae Bursatella leachii Rare5 ✖ ✔ Aplysiidae Dolabrifera dolabrifera Rare (1) ✖ Philinidae Philine angasi Common2 ✖ ✔2 Ostreidae Saccostrea glomerata Abundant ✔ Octopodidae Octopus sp. Rare (1) ✖ Octopodidae Hapalochlaena maculosa Rare (1) ✖ Total number of species 21178 11 8

173 Common on the sea-side of the eastern wall but rare in the pool 174 These species were found in the sand on the bottom of the pool during a cleaning period 175 Only juveniles and young adults 176 Uncommon in the pool but common along the top of the wall at the eastern end and on the sea-side of the wall 177 These species have fluctuating populations and were not always recorded 178 Number of species that were:- Abundant = 5; Common = 11; Uncommon = 2; Rare = 3 One abundant species, four common species and one uncommon species were not recorded on every on every sampling period

Chapter 2 Appendix 2.2 439

Appendix 2.2d: Coalcliff Pool. Habitat: This is a large artificial swimming pool, which is painted. There is a large cement wall on the eastern side, which receives strong wave action. The pool is regularly cleaned.

Inventory: 2/1/98; Tide = 0.2

Other sampling dates: 12/5/95; 23/7/95; 29/11/96; 20/2/97; 28/5/97; 21/8/97; 3/10/97, 28/2/98, 22/6/98

Total number of sampling events:12

Family Species Occurrence Inventory Eggs Neritidae Nerita atramentosa Abundant179 ✔ ✔ Patellidae Cellana tramoserica Common180 ✔ Fissurellidae Montfortula rugosa Common181 ✔ Trochidae Austrocohlea constricta Abundant2 ✔ Littorinidae Nodilittorina unifasciata Abundant ✔ Littorinidae Bembicium nanum Abundant1 ✔ ✔ Muricidae Morula marginalba Common3 ✔ Siphonariidae Siphonaria denticulata Common3 ✔ ✔ Aplysiidae Aplysia juliana Uncommon2 ✔ Chitonidae Chiton pelliserpentis Uncommon3 Ostreidae Saccostrea glomerata Common ✔ Total number of species 11182 9 4

179 Uncommon in the pool but abundant on the eastern wall 180 Uncommon in the pool but common on the eastern wall 181 Only on the sea-side of the eastern wall 182 Only seven species were recorded in the pool. A further four species were recorded on the sea-ward side of the eastern wall. Number of species that were:- Abundant = 4; Common = 5; Uncommon = 2

Chapter 2 Appendix 2.2 440

Appendix 2.2e: Coledale Pool Habitat: Large artificial swimming pool that is not painted. The pool is regularly cleaned and the bottom of the pool is sandy.

Inventory: 3/1/98, Tide = 0.2

Other sampling dates: 10/5/95; 2/10/95; 31/11/96; 23/2/97; 5/3/97; 15/2/98; 22/2/98; 26/3/98 Total number of sampling events: 9 Family Species Occurrence Inventory Eggs Neritidae Nerita atramentosa Abundant ✔ ✔ Patellidae Cellana tramoserica Common183 ✔ Acmaeidae Notoacmaea petterdi Common1 Fissurellidae Montfortula rugosa Common1 ✔ Turbinidae Turbo undulatus Uncommon1 Trochidae Austrocohlea constricta Abundant ✔ Littorinidae Nodilittorina unifasciata Abundant ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ Muricidae Morula marginalba Uncommon1 ✔ Muricidae Dicathais orbita Rare1 Siphonariidae Siphonaria denticulata Uncommon1 ✔ ✔ Siphonariidae Siphonaria funiculata Uncommon1 ✔ Aplysiidae Aplysia sydneyensis Uncommon184 ✔ Aplysiidae Aplysia juliana Uncommon2 ✔ Aplysiidae Aplysia parvula Rare (2) Chitonidae Chiton pelliserpentis Uncommon1 Mopalidae Plaxiphora albida Rare1 Ostreidae Saccostrea glomerata Common ✔ Octopodidae Octopus sp. Rare (1) Total number of species 19185 10 4

183 Only on the sea-side of the northern wall 184 These species were only present in the pool for short periods. 185 Only nine species were recorded in the pool. A further ten species were recorded on the sea-ward side of the northern wall. Number of species that were:- Abundant = 4; Common = 4; Uncommon = 7; Rare = 4

Chapter 2: Appendix 2.2 441

Appendix 2.2f: Towradgi Habitat: A large artificial swimming pool painted with antifouling paint and a cement bottom. Relatively little sand collects on the bottom of the pool. The pool is regularly cleaned. Inventory: 28/1/98; Tide = 0.2 Other sampling dates: 14/8/95; 15/8/95; 9/9/95; 21/9/95; 18/8/96; 23/8/96; 12/9/96; 26/9/96; 3/10/96; 25/10/96; 11/12/96; 28/5/97; 1/7/97; 20/8/97; 15/9/97; 17/10/97; 13/11/97; 27/2/98; 25/4/98 Total number of sampling events: 20

Family Species Occurrence Inventory Eggs Neritidae Nerita atramentosa Common ✔ ✔ Patellidae Cellana tramoserica Common186 ✔ Fissurellidae Montfortula rugosa Common1 ✔ Trochidae Austrocohlea constricta Common ✔ Littorinidae Littorina unifasciata Abundant ✔ Littorinidae Bembicium nanum Common ✔ ✔ Siphonariidae Siphonaria denticulata Uncommon187 ✔ ✔ Siphonariidae Siphonaria funiculata Uncommon2 ✔ Aplysiidae Aplysia sydneyensis Common188 ✖ ✔ Aplysiidae Aplysia juliana Common3 ✔ ✔ Aplysiidae Stylocheilus longicauda Common3 ✖ ✔ Octopodidae Octopus sp. Rare (1) ✖ Octopodidae Hapalochlaena maculosa Rare (1) ✖ Loliginidae Sepioteuthis australis - ✖ ✔189 Total number of species 13190 9 7191

186 Rare in the pool but common on the sea side of the eastern wall. 187 Mostly found on the sea side of the eastern wall. 188 These species have fluctuating populations and are not found all year round. They are most common in Spring to early Summer. 189 One egg mass washed into pool (ie. not attached to substratum). Tangled up in fishing line. 190 Number of species that were:- Abundant = 1; Common = 8; Uncommon = 2; Rare = 2 Three of the species listed as common are only temporarily common. 191 The egg masses of six species were found attached to the pool substratum. The egg mass of one species had been washed in after a storm.

Chapter 2 Appendix 2.2 442

Appendix 2.2g: Wollongong Pools (Wollongong Baths)

Habitat: Two artificial pools that are not painted and are not regularly cleaned. The pools have sandy bottoms.

Inventory: 30/1/98; Tide = 0.1

Other sampling dates: 22/9/95; 19/10/95; 23/7/96; 8/9/96; 17/9/97; 19/9/96; 22/9/96; 18/10/96; 30/1/97; 7/3/97; 5/10/97; 24/11/97; 24/4/98.

Total number of sampling events: 12 Family Species Occurrence Inventory Eggs Neritidae Nerita atramentosa Common ✔ ✔ Patellidae Cellana tramoserica Common ✔ Fissurellidae Montfortula rugosa Common ✔ Trochidae Austrocochlea constricta Common ✔ Trochidae Cantharidella picturata Uncommon ✔ Trochidae Phasianotrochus eximius Rare ✔ Littorinidae Littorina unifasciata Abundant ✔ Littorinidae Bembicium nanum Common ✔ ✔ Muricidae Morula marginalba Common ✔ Siphonariidae Siphonaria denticulata Uncommon ✔ Siphonariidae Siphonaria funiculata Common ✔ Aplysiidae Aplysia sydneyensis Uncommon ✖ ✔ 192 Aplysiidae Aplysia juliana Uncommon1 ✔ ✔ Ostreidae Saccostrea glomerata Uncommon ✔ Total number of species 14193 13 4

192 These species were only recorded on a few occasions 193 Number of species that were:- Abundant = 1; Common = 7; Uncommon = 6; Rare = 0

Chapter 2 Appendix 2.2 443

Appendix 2.2h: Wombarra Pool

Habitat: Large man-made pool not painted with antifouling paint on rock platform. The pool is regularly cleaned. The eastern wall was severely damaged in a storm. The bottom of the pool is sandy.

Inventory: 30/12/97; Tide = 0.2

Other sampling dates: 31/11/96; 21/2/97; 28/5/97; 13/6/97; 7/7/97; 13/9/97; 25/10/97; 1/11/97; 8/11/97; 23/11/97, 17/4/98, 21/6/98.

Total number of sampling events: 12 Family Species Occurrence Inventory Eggs Neritidae Nerita atramentosa Abundant ✔ ✔ Patellidae Cellana tramoserica Common194 ✔ Acmaeidae Notoacmea petterdi Common1 Fissurellidae Montfortula rugosa Common1 ✔ Turbinidae Turbo undulatus Uncommon1 Trochidae Austrocohlea constricta Abundant ✔ Littorinidae Nodilittorina unifasciata Abundant ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ Muricidae Morula marginalba Uncommon1 ✔ Muricidae Dicathais orbita Rare1 Siphonariidae Siphonaria denticulata Uncommon1 ✔ ✔ Siphonariidae Siphonaria funiculata Uncommon1 ✔ Aplysiidae Aplysia sydneyensis Uncommon195 ✔ Aplysiidae Aplysia juliana Uncommon2 ✔ Aplysiidae Aplysia parvula Rare (2) Chitonidae Chiton pelliserpentis Uncommon1 Mopalidae Plaxiphora albida Rare1 Ostreidae Saccostrea glomerata Common ✔ Octopodidae Octopus sp. Rare (1) Total number of species 19196 10 4

194 Only on the sea-side of the northern wall 195 These species were only present in the pool for short periods. 196 Only nine species were recorded in the pool. A further ten species were recorded on the sea-ward side of the northern wall. Number of species that were:- Abundant = 4; Common = 4; Uncommon = 7; Rare = 4

Chapter 2 Appendix 2.3 444

Appendix 2.2i: Wollongong Channel (Flagstaff Point) Habitat: This is a boulder filled channel blocked off by a cement wall. The site is protected from strong swell by the wall. It has a low tidal range but is gently sloping so it is possible to sample the shallow subtidal region. Inventory: 27/2/98; Tide = 0.1 Other sampling dates: 12/10/95; 23/7/96; 19/9/96; 6/12/96; 24/11/97; 28/11/97; 13/12/97; 17/12/97; 6/1/98; 13/1/98; 22/1/98; 23/1/98; 24/4/98. Total number of sampling events: 15

Family Species Occurrence Inventory Eggs Neritidae Nerita atramentosa Abundant ✔ ✔ Patellidae Cellana tramoserica Common ✔ Acmaeidae Notoacmaea petterdi Common ✔ Fissurellidae Montfortula rugosa Abundant ✔ Trochidae Austrocochlea constricta Abundant ✔ Littorinidae Nodilittorina unifasciata Abundant ✔ Littorinidae Nodilittorina pyramidalis Abundant ✔ Littorinidae Bembicium nanum Abundant ✔ ✔ Ranellidae Cabestana spengleri - ✔197 Muricidae Morula marginalba Common ✔ Muricidae Dicathais orbita Rare ✔1 Siphonariidae Siphonaria denticulata Common ✔ ✔ Siphonariidae Siphonaria funiculata Common ✔ Aplysiidae Aplysia sydneyensis Common198 ✔ ✔ Aplysiidae Aplysia juliana Common2 ✔ ✔ Aplysiidae Aplysia dactylomela Rare (1) ✔ Aplysiidae Dolabrifera dolabrifera Rare (2) Aplysiidae Stylocheilus longicauda Common2 ✔ ✔ Pleurobranchidae Pleurobranchea sp. Rare (2) ✔ Elysiidae Elysia australis Common2 ✔ Unidentified Unidentified eggs sp 3199 Rare (3) ✔ Ischnochitonidae Ischnochiton australis Uncommon Chitonidae Onithochiton quercinus Rare Chitonidae Chiton pelliserpentis Common ✔ Mytilidae Trichomya hirsuta - Montacutidae Kellia rotunda Rare (2) Ostreidae Saccostrea glomerata Uncommon ✔ Octopodidae Octopus sp. Rare (1) Idiosepiidae Idiosepius notoides Rare (1) Total number of species 27200 16 11201

197 Hatched egg mass washed up on beach 198 These species have fluctuating populations and are no always observed. 199 White spiralled ribbons flattened against the rock surface. Laid on the underside of boulders at low water mark. 200 Number of species that were:- Abundant = 6; Common = 10; Uncommon = 2; Rare = 9 Four of the species recorded as common are only temporarily common. One of the rare species was only recorded from the presence of egg ribbons 201 The egg masses of nine species were found in the channel attached to the substratum. The hatched egg capsules of a further two species were found washed up.

Chapter 2 Appendix 2.3 445

Appendix 2.3

Breeding Habitats

Summary of the breeding habitats used by intertidal molluscs along the Wollongong Coast, NSW, Australia.

The substrata used for the deposition of egg masses has been listed for each species. The substratum have been classified as:- A = Exposed rock surfaces B = The underside of submerged boulders C = Algae D = Embedded in sand E = Multiple intertidal reef substrata (includes exposed rock, under boulders and/or algae). F = Natural estuarine substrata G = Subtidal (i.e the eggs were only observed in the subtidal region or washed up amongst beach debris).

The deposition of benthic egg masses in artificial habitats, as well as on naturally wave exposed and/or sheltered intertidal reefs, has been recorded for each species of mollusc.

Chapter 2 Appendix 2.3 446

Appendix 2.3: Breeding habitats used by intertidal molluscs along the Wollongong Coast, NSW, Australia. Species Site of deposition Substrate Natural Reefs Artificial Habitats Sheltered Exposed Pools Channel Nerita atramentosa High intertidal rock A ! ! ! ! Bembicium nanum High intertidal rock A ! ! ! ! Bembicium auratum Hard estuarine substrate F Conuber cf. sordidus Free in estuarine waters F Cabestana spenglerii Shallow subtidal caves G Agnewia tritoniformis Under submerged boulders B ! Bedeva hanleyi Under submerged boulders B ! ! Bedeva paivae Amongst oysters on F estuarine boulders Dicathais orbita Swash zone on vertical E ! ! rock walls, overhangs, & Pyura stolonifera tests. Ergalatax contracta Under submerged boulders B ! ! Lepsiella reticulata Submerged algal fronds C ! ! Morula marginalba Under submerged boulders B ! ! Nassarius jonasii Unknown202 Mitra badia Under submerged boulders B ! Mitra carbonaria Under submerged boulders B ! Conus paperliferus Under submerged boulders B ! Unidentified Cerithiidae Attached to algae in a pool B ! Unidentified sp. 1 Caulerpa filiformis fronds E ! ! and under boulders Unidentified sp. 2 Under submerged boulders B ! Unidentified sp. 3 Under submerged boulders B ! Unidentified sp. 4 Under submerged boulders B ! Unidentified sp. 5 Subtidal G Unidentified sp. 6 Subtidal G Unidentified sp. 7 Subtidal G Unidentified sp. 8 Subtidal G Unidentified sp. 9 Subtidal G

202 The eggs were only observed in the aquaria but the adults were found in naturally sheltered habitats and artificial pools

Chapter 2 Appendix 2.3 447

Appendix 2.3: Breeding habitats continued. Species Site of deposition Substrate Natural Habitats Artificial Habitats Sheltered Exposed Pools Channel Siphonaria denticulata Mid-low intertidal rock A ! ! ! ! Siphonaria zelandica Mid-low intertidal rock A ! ! Salinator fragilis Estuarine mud or sand flats F Salinator solidus Estuarine mud or sand flats F Unidentified sp. 14 Estuarine mud F Aplysia dactylomela Submerged algae & boulders E ! Aplysia parvula Submerged algae & boulders E ! Aplysia sydneyensis Submerged boulders & algae E ! ! ! Aplysia juliana Submerged boulders & algae E ! ! ! ! Dolabrifera dolabrifera Under submerged boulders B ! ! Stylocheilus longicauda Submerged boulders & algae E ! ! ! Bursatella leachii Submerged rock & algae E ! Philine angasi Subtidal sand D ! Berthellina citrina Under submerged boulders B ! Pleurobranchus peroni Under submerged boulders B ! Pleurobranchea sp. Under submerged boulders B ! ! ! Bullina lineata Shallow sand D ! ! ! Hydatina physis Shallow sand D ! ! Oxynoe viridis Caulerpa filiformis fronds C ! ! Elysia australis Under submerged boulders B ! ! ! Dendrodoris fumata Under submerged boulders B ! Dendordoris nigra Under submerged boulders B ! Doriopsilla miniata Under submerged boulders B ! Doriopsilla carneola Under submerged boulders B ! Discodoris fragilis Under submerged boulders B ! Platyodoris galbannus Under submerged boulders B ! Jorunna pantherina Under submerged boulders B ! Rostangia bassia Under submerged boulders B ! ! Hypselodoris bennetti Under submerged boulders B !

Chapter 2 Appendix 2.3 448

Appendix 2.3: Breeding habitats continued. Species Site of deposition Substrate Natural Habitats Artificial Habitats Sheltered Exposed Pools Channel Plocamperus imperialis On submerged boulders A ! Goniodoris sp. Under submerged boulders B ! ! Austraeolis ornata Under submerged boulders B ! ! Aeolidella foulisi Under submerged boulders B ! ! Unidentified sp. 10 Under submerged boulders B ! ! Unidentified sp. 11 Under submerged boulders B ! Unidentified sp. 12 Rock pool wall A ! Unidentified sp. 13 Subtidal G Sepioteuthis australis Subtidal G Octopus sp. Subtidal caves G Total Number of species 65 44 22 9 11 203

203 The egg masses from 49 species were found on natural intertidal reefs and/or in artificial habitats. The number of species found attaching egg masses to the different intertidal substratum was:- A) Exposed rock = 6 B) Under boulders = 29 C) Algae = 3 D) Sand = 3 E) Multiple substratum = 8 The egg masses from 6 species were found in natural estuarine environments (F). The egg masses from a further 9 species were found amongst beach debris or in the subtidal zone (G). The eggs from one species were deposited in aquaria but not observed in the field.

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Chapter 4: Appendix 4 Antimicrobial Assays and epibiosis 449

Chapter 4: Appendix 4 Antimicrobial Assays and Epibiosis

Appendix 4.1

Alternative methods for the Zone of Inhibition assay

4.1.1 Bacterial lawns Alternative methods for producing an even lawn of bacteria on agar plates in the Zone of Inhibition assay were trailed using the egg ribbons of Aplysia juliana and Dicathais orbita. Bacterial cultures were inoculated directly into the cooled but molten agar

(~400C) before pouring the plates rather than spreading the cultures on the surface of the agar. This method produced an even distribution of the microorganism throughout the surface of the agar but the zones of inhibition were more difficult to detect than when the surface was spread with the microbial culture.

4.1.2 Microbial Growth Phase The eggs of Aplysia juliana were tested against Staphylococcus aureus and

Escherichia coli at three different stages of growth; stationary, early exponential and exponential growth phases. Zones of inhibition around the egg ribbons were similar for the three different growth phases and it was decided that cultures in early exponential growth phase would be the most appropriate for general use.

4.1.3 Containing specimens on the agar surface Various methods for containing the samples on the agar surface were trialed using the egg masses of Aplysia juliana and Dicathais orbita. These include the agar cup method, the filter paper disk diffusion method and direct placement onto the agar surface (refer to Spooner and Skyes, 1972). The egg ribbons of Aplysia juliana consistently produced the largest and clearest zones of inhibition when placed directly Chapter 4: Appendix 4 Antimicrobial Assays and epibiosis 450 onto the agar surface. The egg capsules of Dicathais orbita only produced unambiguous zones of inhibition when placed directly onto the agar surface.

The use of inoculated sterile filter paper disks (0.9mm diameter) was also compared to direct inoculation of the agar with the intracapsular fluid from the egg mass of

Dicathais orbita. The egg capsules were crushed using a mortar and pestle and 20µl of the intracapsular fluid was pipetted onto the filter paper disks or directly onto the agar. Impregnated filter paper disks were then transferred to the agar using sterile forceps. Small zones of inhibition were apparent around the directly inoculated samples but no clear zone was visible around the filter paper disks. It was decided that the most appropriate method for detecting a zone of inhibition around samples of egg material was to place them directly on the surface of the agar.

Appendix 4.2

Methods for the Fluorescein Diacetate assay

4.2.1 Preliminary method development Trial plates with the four human pathogens were run using Milli Q water and acetone with FDA, to determine the incubation time and to assess the effects of acetone on the cultures. Cultures of the Gram negative bacteria, Escherichia coli and Pseudomonas aeruginosa were found to rapidly hydrolyse Fluorescein Diacetate (FDA), producing clearly visible results within 2 hrs. Staphylococcus aureus typically took 3.5- 4 hrs before the production of FDA was clearly visible. There was no difference in FDA production between the bacterial cultures incubated with acetone or Milli Q water. On the other hand, very inconsistent results were obtained using Candida albicans

(ACM4581). On several occasions no FDA hydrolysis could be detected after a 24hr Chapter 4: Appendix 4 Antimicrobial Assays and epibiosis 451 incubation period. Two additional strains of C. albicans (AMMRL 36.42, AMMRL

36.70) were tested with similar results. The inconsistent growth could not be directly attributed to the acetone. Some cultures with acetone appeared to hydrolyse more

FDA than those with Milli Q water, whilst others did not hydrolyse any FDA.

4.2.2 Alternative growth conditions for Candida albicans. Three different broths and incubation temperatures were trialed for Candida albicans

(ACM4581) in an attempt to optimise the growth rate. These trials were run with pure broth cultures in the absence of acetone. The production of fluorescence was clearly visible after 4.5 hrs at 370C using Sabourad Liquid Medium and the malt, yeast, peptone, glucose broth. By comparison, after an equivalent period of time, fluorescence was only just detectable in these broths at an incubation temperature of

280C. Using the peptone, glucose broth, fluorescence was only just detectable after an incubation period of 4.5 hrs at 370C. At 280C cultures incubated in peptone, glucose did not fluoresce after 5hrs. It was decided that Sabourad Liquid Medium and an incubation temperature of 370C were the most appropriate conditions in which to grow Candida albicans (ACM4581). However, inconsistent growth was still obtained when acetone was added to any of the wells on the plate. Consequently, it was inferred that acetone affects the growth of Candida albicans even if the cells are not directly incubated with this solvent. Similar problems with using Candida albicans in the FDA assay have been experienced by a research group at Macquarie University

(Dr Duncan Veal, pers. comm.).

Chapter 4: Appendix 4 Antimicrobial Assays and epibiosis 452

4.2.3 Alternative methods for transferring extracts An alternative method for transferring the extracts to the microtitre plates was trialed using extracts from Dicathais orbita and Aplysia juliana. This involved dissolving the extracts in ethanol and transferring them to the microtitre plate before the microbial cells were introduced. The majority of the solvent was then dried down in a fume hood and the remaining solvent was removed by placing the plates in a vacuum

(0.1mm Hg, 30 min – 1 hr). The microbial cultures were then added to the plates and the plates were agitated before incubation. This method produced inconsistent results and in general antimicrobial activity was not detected at the same concentration as seen using the acetone method. This could be because the extracts were not solublised in the broth and therefore the active components were not in contact with the microbial cells. Nevertheless, this method has proved effective for the research group at Macquarie University (Dr Duncan Veal, personal communication).

Appendix 4.3

Microfauna associated with molluscan egg masses

4.3.1 Overnight incubations on marine and nutrient agar. The microorganisms found associated with the egg masses of 17 molluscs and three polychaetes are summarised in Table 4.11. The samples were placed marine agar and incubated overnight at 250C. Observations were also made from samples that were placed on Nutrient agar spread with Escherichia coli or Staphylococcus aureus and used in the Zone of Inhibition assay. Chapter 4: Appendix 4 Antimicrobial Assays and epibiosis 453

Table 4.11: Description of the microfauna found associated with the egg masses of 17 molluscs, three polychaetes and some intertidal substrate after one night incubation at 370C on Nutrient agar or 250C on Marine agar.

Species Agar Description of epibiotic organisms Phylum: Mollusca, Class: Gastropoda Nerita atramentosa Nutrient Surrounded by cream colonies Bembicium nanum Nutrient Surrounded by cream colonies Marine Surrounded by cream colonies Conuber cf. sordidus Nutrient Surrounded by cream colonies, white filamentous fungus Marine Surrounded by cream colonies Dicathais orbita Marine Scattered white colonies Agnewia tritoniformis Marine Surrounded by white colonies Mitra carbonaria Marine Surrounded by white colonies Conus paperliferus Nutrient White filamentous fungi (Hatched capsules) Marine Scattered white colonies Salinator fragilis Nutrient Surrounded by cream colonies, white filamentous fungi Marine Surrounded by white colonies Salinator solidus Nutrient Surrounded by cream colonies SDA Surrounded by cream colonies Unidentified sp. 14 Nutrient Surrounded by cream colonies, white filamentous fungi Aplysia juliana Marine Scattered white colonies Aplysia sydneyensis Nutrient Surrounded by cream colonies (not crushed) Aplysia sydneyensis Marine Surrounded by white colonies Dolabrifera dolabrifera Marine Surrounded by white colonies Pleurobranchus peroni Marine Surrounded by white colonies Bullina lineata Nutrient Surrounded by cream colonies Hydatina physis Nutrient Surrounded by cream colonies Philine angasi Nutrient Surrounded by cream colonies Phylum: Annelidia, Class: Polychaeta Unidentified sp. 1 Nutrient Scattered cream colonies Unidentified sp. 2 Nutrient Surrounded by cream colonies, white filamentous fungi Unidentified sp. 3 Marine Surrounded by white colonies Intertidal substrate Intertidal sand Nutrient Surrounded by cream colonies Estuarine mud Nutrient Surrounded by cream colonies, white filamentous fungi Marine Surrounded by white colonies Chapter 4: Appendix 4 Antimicrobial Assays and epibiosis 454

4.3.2 Microfauna present after one week at 40C The microfauna associated with the egg masses of Dicathais orbita and

Aplysia juliana were observed on Nutrient agar after one week at 40C. The only microbes observed around the egg ribbons of Aplysia juliana were the same white colonies that were present after one nights incubation.

Conversely, orange, yellow and white colonies were found on the intracapsular fluid of the well developed larvae from Dicathais orbita egg capsules. These colonies were exclusively associated with the intracapsular fluid or egg capsules but were not found anywhere else on the agar. Some colonies were also observed on the intracapsular fluid at an intermediate stage of development but none were observed on the fresh eggs. This suggests that the intracapsular fluid from freshly laid D. orbita capsules is able to suppress microbial growth for at least one week at 40C.

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Chapter 6: Appendix 6 455

Chapter 6: Appendix 6

Appendix 6.1: Composition of volatile organic compounds found in the extracts of 23 marine molluscs using gas chromatography/ mass spectrometry (GC/MS). Appendix 6.2: Mass spectra of the brominated compounds found in the egg masses of Muricids. Appendix 6.3: Brominated compounds detected in the egg mass of Dicathais orbita by GC/MS in chemical ionisation mode. Appendix 6.4: Mass spectra of nonbrominated indoles found in the egg masses of Muricids. Appendix 6.5: Mass spectra of sulfoxides and sulfides found in the egg masses of Muricids. Appendix 6.6: Mass spectra of chlorinated compounds found in the egg masses of Aplysiidae. Appendix 6.7: Mass spectra of sulfur and hexathiepane found in the egg mass of Salinator fragilis and estuarine mud. Appendix 6.8: Composition of volatile organic compounds found in solvent controls and extracts taken from seawater, estuarine mud and intertidal pebbles. Chapter 6: Appendix 6.1 456 Appendix 6.1 Appendix 6.1a Agnewia tritoniformis Fraction 1 R.t. Area Identity M+ / F+ % fraction 1 % total 21.767 4.5 octyne 0.300782 0.291621 26.683 5.5 unknown 0.367622 0.356425 27.233 11.7 unknown 193 0.782033 0.758214 28.333 30.4 unknown 141 2.03195 1.97006 30.517 3 unknown 162 0.200521 0.194414 30.583 7.3 unknown 147 0.487935 0.473074 32.7 5.8 unknown 221 0.387675 0.375867 33.083 5.4 unknown 302 0.360938 0.349945 33.117 8.5 unknown 304 0.568144 0.550839 34 4.7 alkene 0.31415 0.304582 34.533 13.5 unknown 0.902346 0.874862 34.583 8.8 unknown 280 0.588196 0.570281 34.783 3 unknown 149 0.200521 0.194414 34.9 2.4 unknown 181 0.160417 0.155531 35.067 4.5 enal 0.300782 0.291621 35.85 7.5 alkane C14? 198 0.501303 0.486035 36.05 4.3 unknown 234 0.287414 0.27866 36.283 13.5 unknown 203 0.902346 0.874862 37.267 3.9 fatty acid ethyl ester 0.260678 0.252738 37.45 19.1 alkane 1.276653 1.237768 37.883 28.9 aldehyde 1.931689 1.872853 37.967 7.7 tyrindoleninone 255, 257 0.514671 0.498996 41.6 5.3 fatty acid 0.354254 0.343464 41.8 9.8 acid? 0.655036 0.635085 42 8.1 ene one 0.541408 0.524917 42.167 14.2 aldehyde 0.949134 0.920226 42.417 10.9 fatty acid ethyl ester 0.728561 0.70637 43.167 439.2 aldehyde 29.35633 28.46219 43.683 6.8 unknown 217 0.454515 0.440671 44.583 34.7 alcohol 2.319364 2.24872 49.983 6.7 unknown 315 0.447831 0.434191 59.5 63.2 trimethyl tridecatrienenitrile 231 4.224317 4.095652 60.95 28.1 unknown 364 1.878217 1.82101 64.567 483.8 cholesterol 386 32.33741 31.35247 64.633 23.7 cholestanol 388 1.584119 1.535869 65.167 89.4 cholestadienol 384 5.975536 5.793532 65.517 13.8 sterol 369 0.922398 0.894304 66.133 13.4 sterol 383 0.895662 0.868382 66.25 11.6 methylcholestenol 400 0.775349 0.751734 68.067 24 sterol 414 1.604171 1.555311 68.433 9.7 sterol 0.648352 0.628605 71.617 12.6 sterol ? 0.84219 0.816538

Chapter 6: Appendix 6.1 457

Appendix 6.1a Contd. Agnewia tritoniformis Fraction 2 R.t. Area Identity M+ / F+ % fraction 1 % total 30.583 5.3 unknown 147 11.2766 0.343464 30.8 3.4 unknown aromatic 133 7.234043 0.220336 33.067 7.4 phosphoric acid ester 306 15.74468 0.479554 36.317 1.4 alcohol 2.978723 0.090726 41.583 13.9 C17 acid 256 29.57447 0.900784 41.683 8.4 amide 110 17.87234 0.544359 46.56 7.2 amide 128 15.31915 0.466593

Chapter 6: Appendix 6.1 458

Appendix 6.1b Ceratostoma erineceum Fraction 1 R.t. Area Identity fragments % fraction % total 9.633 628994 dimethyl trisulfide 126, 111 2.491899 2.109234 10.317 68932 phenol 142, 94 0.273089 0.231153 10.817 58194 ene ol/ thiol 111, 97 0.230548 0.195145 13.1 48758 methyl methane thiosulphonate 154, 126 0.193166 0.163502 14.9 253485 unknown 121, 56 1.004237 0.850023 17.6 30608 unknown 150, 128 0.12126 0.102639 17.733 67891 sulfated 155, 153 0.268965 0.227662 17.983 108307 ether? 57, 43 0.429082 0.363191 18.55 1449077 brominated unknown 219, 217 5.740838 4.859256 18.85 32972 sulphide 194, 158 0.130626 0.110567 20.017 69113 C6 dioic acid di me ester 143, 132 0.273806 0.23176 21.25 332632 unsaturated oxygenated 91, 79 1.317795 1.11543 21.683 33584 unknown 215, 175 0.13305 0.112619 22.033 114583 dibutyl formamide 152, 157 0.453946 0.384236 23.483 72497 unknown 153, 116 0.287213 0.243107 23.617 59621 ester 129, 109 0.236202 0.19993 23.833 231494 tetrabutyl urea 156, 106 0.917115 0.776279 24.083 82806 indene dione (closest) 161, 146 0.328054 0.277677 24.25 48408 unknown 172, 145 0.191779 0.162329 24.5 58338 ester? 187 0.231119 0.195627 24.717 46531 unknown 77, 148 0.184343 0.156035 24.2 68907 vanillin 151, 152 0.27299 0.231069 25.533 61085 alkane 0.242002 0.204839 25.85 84214 unknown 127 0.333632 0.282399 26.683 183507 pyrazole methyl phenyl (closest) 158, 252 0.727003 0.615362 27.017 237029 C11 diene one 194 0.939043 0.79484 27.35 132998 unknown 165, 180 0.526901 0.445988 27.667 19185 isoindol diene 76, 147 0.076006 0.064334 27.8 61960 dibromo pyrazole/ imidazole 199, 226 0.245468 0.207773 27.883 22708 unknown 163 0.089963 0.076148 27.967 162501 alkene 0.643784 0.544922 28.45 404526 unknown 141, 181 1.602619 1.356515 28.767 133197 alkane 0.527689 0.446656 29.217 164324 urea 72, 170 0.651006 0.551035 30.25 69036 brominated 196, 198 0.273501 0.231502 30.317 159012 methyl thioindolone 50, 177 0.629961 0.533222 30.867 131165 unknown 127 0.519639 0.439842 30.933 53759 C6 dioic acid di me ester 143 0.212978 0.180272 31.317 1509280 alkene 57, 159 5.979346 5.061137 31.867 184586 alkane 0.731278 0.618981 32.4 893150 brominated 226, 239 3.538411 2.99504 32.633 95235 brominated unknown 229, 293 0.377294 0.319356 33.5 309476 tri bromo pyrazole 227, 306 1.226057 1.03778 34.55 41429 brominated 226, 344 0.16413 0.138926 34.9 113705 unknown 105, 135 0.450467 0.381292 35.117 269202 phenoxy benzene (closest) 103, 198 1.066503 0.902727 35.183 277836 aldehyde 1.100709 0.93168 37.583 320014 alkane 1.267806 1.073117 37.75 153677 alkane 0.608825 0.515332 38 137062 aldehyde 0.543001 0.459616 38.1 265655 tyrindoleninone 255, 257 1.052451 0.890833 38.25 6072980 isopropyl myristate 24.05945 20.3648 38.3 341446 brominated 255, 257 1.352714 1.144986 Chapter 6: Appendix 6.1 459

Appendix 6.1b: Ceratostoma erineceum, Fraction 1 continued. R.t. Area Identity fragments % fraction % total 38.517 149427 aldehyde 0.591988 0.50108 38.633 362587 alkane 1.436468 1.215879 40.2 361553 alkane 1.432372 1.212412 40.65 114973 aldehyde 0.455491 0.385544 41.8 324920 unknown brominated 273, 271 1.287242 1.089569 42.7 487155 bromoisatin 197, 225 1.929972 1.633599 42.85 172586 bromoisatin 0.683738 0.57874 43.217 518056 aldehyde 2.052393 1.737221 45.067 94161 unknown 244, 259 0.37304 0.315754 45.117 175942 brominated 257, 244 0.697033 0.589994 45.2 106487 brominated 257, 244 0.421872 0.357088 45.3 662507 alcohol? 2.624668 2.221615 45.6 91977 brominated 219, 217 0.364387 0.308431 47.567 129402 alkene? 0.512655 0.43393 50.883 317968 diene ol? 1.2597 1.066257 51.417 208618 C6 dioic acid di octyl ester 0.826486 0.699568 59.65 725242 amide 2.873206 2.431987 60.683 97582 sterol 368 0.386593 0.327226 61.083 99806 sterol 364 0.395404 0.334684 61.417 78575 sterol 301 0.311292 0.263489 61.733 48611 sterol 366, 383 0.192583 0.163009 63.867 132102 sterol 300, 385 0.523351 0.442984 64.617 1925649 cholesterol 386 7.628884 6.457366 64.75 117176 sterol 263, 388 0.464219 0.392932 64.9 134219 sterol 430 0.531738 0.450083 65.283 230138 sterol 351, 404 0.911742 0.771732 65.683 55447 sterol 350, 368 0.219666 0.185933 66.3 61627 sterol 300, 314 0.244149 0.206657 68.233 98644 sterol 397, 415 0.3908 0.330787 68.6 51392 sterol 314, 392 0.203601 0.172335 71.833 76357 sterol 392, 419 0.302505 0.256051

Fraction 2 R.t. Area Identity fragments % fraction % total 5.8 555660 acid 12.13388 1.86332 10.417 366049 acid 7.993366 1.227489 15.583 157773 acid 3.445269 0.529067 17.55 151468 acid 3.307588 0.507925 18.567 1024247 unknown 105 22.36635 3.434654 21.017 143250 acid 3.128132 0.480367 30.633 128757 acid 2.81165 0.431767 39.633 326905 alcohol 354, 336 7.138583 1.096225 43.083 142937 alcohol 3.121297 0.479317 44.65 121696 cycloalkane 392 2.65746 0.408089 55.7863 1460668 unknown 31.89642 4.898124 Chapter 6: Appendix 6.1 460

Appendix 6.1c Dicathais orbita fresh eggs 1 Fraction 1 Retention time Area Identity M+ % fraction 1 % total 27.783 3.1 unknown 1.142647 1.011749 30.55 1.6 unknown 0.589753 0.522193 32.267 13.7 unknown 5.04976 4.471279 37.967 93.9 tyrindoleninone 255, 257 34.61113 30.64621 40.017 4.2 C14 ketone 212 1.548102 1.370757 40.483 4.9 alcohol or aldehyde 1.806119 1.599217 41.55 3.2 unknown (brominated) 271, 273 1.179506 1.044386 42.117 1.9 bromoisatin 0.700332 0.620104 43.033 13.6 C18 aldehyde 268 5.012901 4.438642 44.35 4.1 unsaturated ketone? 1.511242 1.33812 59.467 6.7 alkene 2.469591 2.186684 63.617 4.4 unknown sterol 1.621821 1.436031 64.333 89.4 cholesterol 32.95245 29.17755 64.7 6.5 unknown sterol 2.395872 2.12141 64.983 11.1 unknown sterol 4.091412 3.622715 67.95 9 unknown sterol 3.317361 2.937337

Fraction 2 Retention time Area Identity M+ % fraction 1 % total 14.867 3.1 C9 aldehyde 8.831909 1.011749 17.3 2.4 heptane 100 6.837607 0.78329 18.433 4.4 unknown 12.53561 1.436031 18.633 6.8 octanal 228 19.37322 2.219321 37.95 10.2 tyrindoleninone 255, 257 29.05983 3.328982 41.567 4.4 C16 fatty acid me ester 12.53561 1.436031 42.2 3.8 bromo indol-2,3 dione 225, 227 10.82621 1.240209 Chapter 6: Appendix 6.1 461

Appendix 6.1c Dicathais orbita fresh eggs 2 Fraction 1 R.t. Area Identity fragments % fraction % total 17.8 50363 unknown 107, 83 5.672039 2.692202 28.417 45823 unknown 141 5.16073 2.449512 30.817 18683 unknown 127 2.104138 0.998718 32.283 26239 brominated 241, 226 2.955119 1.402631 32.583 11139 brominated 231, 199 1.254509 0.595446 33.5 11631 di brominated 302, 304, 306 1.30992 0.621746 36.083 7126 brominated 257, 226 0.802552 0.380927 37.983 9441 unknown 231, 199 1.063275 0.504678 38.167 119973 tyrindoleninone 257, 242 13.51174 6.413271 39.167 7537 brominated 257, 242 0.848841 0.402898 40.15 22406 ketone 2.523434 1.197734 41.733 9648 brominated 226, 172 1.086588 0.515743 42.633 55361 bromoisatin 6.23493 2.959375 43.167 42519 aldehyde 4.788623 2.272894 45-46 3942 tyriveridin 259, 257, 255 0.44396 0.210723 63.833 25850 sterol 2.911308 1.381836 64.533 281256 sterol 31.67593 15.03481 64.7 20584 sterol 2.318235 1.100337 64.85 40776 sterol 4.592321 2.17972 65.217 65793 sterol 7.409814 3.517028 66.267 11827 sterol 1.331994 0.632224

Fraction 2 R.t. Area Identity fragments % fraction % total 30.45 14686 acetaldehyde 1.494329 0.785054 30.8 13202 fatty acid 1.343329 0.705726 30.9 12100 unknown 127 1.231199 0.646817 36.533 17913 unknown 95, 73 1.822683 0.957557 37.55 17664 brominated 213, 211 1.797347 0.944246 37.58 6451 aromatic 105 0.656402 0.344844 38.017 28499 aldehyde 2.899829 1.523441 38.2 84982 tyrindoleninone 257, 242 8.647086 4.542794 39.717 24266 unsaturated oxygenated 81, 67 2.469113 1.297162 40.667 18700 aldehyde 1.902762 0.999626 41.867 91091 C16 acid 9.268688 4.869356 42.667 28386 bromoisatin 227, 199 2.888331 1.517401 43.217 183036 aldehyde 18.62427 9.784364 44.233 24603 me ester 2.503404 1.315177 46.633 83620 acid 8.508499 4.469987 49.133 37504 amide 72, 59 3.816106 2.004812 50.45 46951 ene one 4.777357 2.509811 50.6 45135 alcohol/ene 4.592575 2.412734 50.817 18216 alcohol 107, 69 1.853514 0.973754 50.967 12783 alkane 1.300695 0.683327 54.133 53977 unknown 364, 192 5.492266 2.885392 54.233 73121 eneol 149, 109 7.440205 3.908753 59.667 45896 unknown 261, 173 4.670008 2.453414

Chapter 6: Appendix 6.1 462

Appendix 6.1c Dicathais orbita frozen eggs Fraction 1 R. t. Area Identity fragments % fraction % total 28.417 74825 unknown 141 4.789125 4.320519 32.4 218308 brominated 241, 226 13.97266 12.60547 36.1 12135 brominated 257, 226 0.776693 0.700695 37.983 16911 aldehyde 1.082377 0.976469 38.3 309579 tyrindoleninone 257, 242 19.8144 17.8756 39.2 26214 brominated 200, 198 1.67781 1.51364 41.717 24785 acid + brominated 226, 198 1.586348 1.431127 42.6 28312 bromoisatin 227, 197 1.812091 1.634782 43.167 105663 aldehyde 6.762891 6.101156 45.05 5717 tyriverdin? 259, 257 0.365913 0.330109 58.383 72966 polyunsaturated oxy. 4.670141 4.213178 63.817 33833 sterol 300 2.165459 1.953573 64.583 500638 sterol 32.04301 28.90767 64.7 35040 sterol 2.242712 2.023268 64.867 44029 sterol 2.818047 2.542307 65.2 53439 sterol 3.420328 3.085656

Fraction 2 R. t. Area Identity fragments % fraction % total 37.933 11172 alkane 6.592784 0.64509 39.683 12167 alkene 7.17995 0.702543 40.617 8062 aldehyde 4.757521 0.465513 41.717 25242 acid 14.89573 1.457515 42.583 14349 bromoisatin 227, 197 8.467585 0.828535 43.167 65456 aldehyde 38.62668 3.779538 46.5 15489 unknown 9.140318 0.89436 59.55 17521 unknown 229, 163 10.33944 1.011692 Chapter 6: Appendix 6.1 463 Appendix 6.1c Dicathais orbita Freeze dried 1 Fraction 1 Fraction 1 R. t. Area Identity fragments % fraction % total 13.117 73363 methyl methane thiosulphonate 126, 79 3.474677 2.521216 17.833 100634 unknown 64, 42 4.766307 3.45842 22.1 18571 unknown 141, 153 0.879574 0.638217 28.417 73685 unknown 141, 181 3.489927 2.532282 30.85 14607 unknown 127 0.691828 0.501989 31.267 24212 unknown 1.146748 0.832077 32.417 226785 brominated 224, 239 10.74117 7.793765 33.5 61575 di bromo 225, 306 2.916364 2.116106 34.517 15007 brominated 255, 224 0.710773 0.515735 35.133 13797 alcohol 213, 198 0.653464 0.474152 36.083 8811 brominated 226, 170 0.417314 0.302802 37.95 16072 aldehyde 151, 138 0.761215 0.552335 38.233 266146 tyrindoleninone 257, 242 12.60542 9.146458 39.15 7796 brominated 258, 198 0.36924 0.26792 39.733 5032 brominated 242, 212 0.23833 0.172931 40.617 32642 alkane 1.546016 1.121785 41.783 94348 acid 256 4.468585 3.242393 42.7 35376 bromoisatin 255, 199 1.675506 1.215743 43.167 110348 aldehyde 5.226389 3.792254 43.283 28141 acid 1.332836 0.967103 44.15 28666 unknown 1.357702 0.985145 45-46 13920 tyriverdin 257, 242 0.65929 0.478379 46.55 93634 acid 284 4.434768 3.217856 49.55 24832 unknown 1.176113 0.853384 50.367 45468 unsaturated acid/ ene ol? 2.153491 1.562568 50.483 45141 ene al 2.138004 1.55133 50.883 18011 unsaturated oxygenated 0.853051 0.618972 59.533 27625 unknown 218, 199 1.308397 0.949369 60.65 19894 sterol 213, 250 0.942235 0.683683 61.033 27993 unknown 251 1.325827 0.962016 63.8 29623 unknown 301, 273 1.403028 1.018033 64.55 402732 sterol 19.07451 13.84042 64.867 38106 disulphide 164, 165 1.804807 1.309563 65.2 47494 sterol 271 2.249448 1.632194 65.35 21275 unknown 264, 210 1.007643 0.731143

Chapter 6: Appendix 6.1 464

Appendix 6.1c contd. Dicathais orbita Freeze dried 1 Fraction 2 R. t. Area Identity fragments % fraction % total 30.783 24176 fatty acid 199, 181 3.027813 0.83084 37.533 20747 brominated 242, 240 2.598364 0.712998 37.583 21254 brominated 232, 230 2.661861 0.730422 37.95 26382 aldehyde 82 3.304094 0.906652 38.133 46252 tyrindoleninone 5.792622 1.589511 39.683 33601 alcohol 97, 83 4.208205 1.154743 41.85 126066 fatty acid 15.78856 4.332424 43.2 47639 aldehyde 5.96633 1.637177 44.2 21321 acid 2.670252 0.732724 45-46 15028 tyriverdin 257, 244 1.882114 0.516457 49.583 31866 unsaturated 3.990913 1.095117 50.483 28233 unsaturated 3.535914 0.970264 59.65 312086 unknown 217, 173 39.08579 10.72525 64.917 43813 sterol 5.48716 1.505691

Chapter 6: Appendix 6.1 465

Appendix 6.1 Dicathais orbita Freeze dried 2 Fraction 1 R. t. Area Identity fragments % fraction % total 13.117 23181 thio sulphonate 126, 176 3.64939 2.880024 17.75 25581 unknown 136, 108 4.027223 3.178202 28.433 27400 unknown 141, 113 4.313588 3.404196 32.333 103455 brominated 241, 226 16.28694 12.85333 32.567 17214 brominated 231, 199 2.710004 2.13868 38.167 65306 tyrindoleninone 257, 242 10.28114 8.113665 41.733 46834 C 16 acid 227, 199 7.373088 5.818691 42.6 45912 bromoisatin 7.227937 5.704141 43.167 61617 ketone 9.700379 7.655341 64.533 218702 sterols 34.43031 27.1717

Fraction2 R. t. Area Identity fragments % fraction % total 38.183 24516 tyrindoleninone 14.44778 3.045886 39.717 18681 ene ol 97, 82 11.00909 2.320941 41.817 44323 C 16 acid 26.12045 5.506722 43.233 82167 aldehyde 48.42268 10.20849 Chapter 6: Appendix 6.1 466

Appendix 6.1c Dicathsi orbita Hatching eggs Fraction 1 R. t. Intensity Identity fragments % fraction % total 4.45 518638 di oxy 2.199958 0.657855 10.583 2000318 alcohol 186 8.484947 2.53726 21.983 119808 alkane 0.508201 0.151968 22.417 130114 unknown 243, 185 0.551917 0.16504 23.35 145085 ketone 0.615421 0.18403 23.867 960326 unknown 169 4.07351 1.218105 27.217 268643 dione 169 1.13953 0.340754 28.317 125231 unknown 141 0.531205 0.158847 28.417 77011 alkene 0.326665 0.097683 28.667 150527 alkane 0.638505 0.190933 29.9 150885 unknown 227 0.640024 0.191387 30.583 58841 unknown 127 0.249592 0.074636 33.967 81138 alkene 0.344171 0.102918 34.25 144084 hexadioic acid dipropenyl ester 215, 169 0.611175 0.18276 34.45 81372 ene one/ ene al 0.345164 0.103215 35.05 106822 aldehyde 0.453117 0.135496 36.4 277372 C14 acid 228 1.176556 0.351827 36.817 124261 alkene 0.52709 0.157616 37.85 226861 aldehyde 0.962299 0.287757 39.067 293466 C15 acid 242 1.244824 0.372241 39.517 401902 alcohol 1.704788 0.509784 40.517 383713 aldehyde 1.627633 0.486713 40.65 129168 C16 acid 256 0.547905 0.16384 41.917 1519222 C16 acid 256 6.444234 1.927025 43.067 583965 aldehyde/ alcohol 2.477062 0.740718 43.35 937213 C17 acid 270 3.975469 1.188788 43.483 318200 C17 acid 270 1.34974 0.403614 44.217 744890 C17 acid 270 3.159674 0.94484 44.567 260027 alkene 1.102982 0.329826 46 375739 alcohol/ aldehyde 1.593809 0.476598 46.117 206306 unsaturated 264 0.875109 0.261684 46.75 2596923 C18 acid 284 11.01563 3.294011 47.15 169222 C18 acid oxy me ester 0.717806 0.214646 48.2 145611 acid 284 0.617653 0.184697 48.683 257449 acid 1.092047 0.326556 49.917 296553 eneol 1.257918 0.376156 50.583 1504592 unsaturated oxygenated 292 6.382177 1.908467 50.7 1536204 unsaturated oxygenated 292 6.516269 1.948565 51.017 724646 acid 312 3.073803 0.919162 54.383 299484 ene ol 1.270351 0.379874 54.533 267365 ene ol 1.134109 0.339133 54.883 317690 unknown 103 1.347577 0.402967 63.65 24157 sterol 384 0.102469 0.030641 64.467 2844299 cholesterol 386 12.06494 3.60779 64.567 395416 sterol 390, 388 1.677275 0.501557 64.967 44486 sterol 398, 384 0.188701 0.056427 66.533 249658 sterol 384 1.058999 0.316673 Chapter 6: Appendix 6.1 467

Appendix 6.1c Dicathais orbita Hatching eggs continued. Fraction 2 R. t. Intensity Identity fragments % fraction % total 10.45 520102 C8 dione 142 0.941143 0.659712 10.7 2209440 alcohol? 155, 186 3.99806 2.802517 11.067 357579 fatty acid 0.647052 0.453563 13.383 651942 oxy acid me ester or ketone 1.179712 0.826942 17.283 363622 unsaturated amine 0.657987 0.461229 23.333 324994 unknown 111, 137 0.588088 0.412232 24 1013539 unknown 169, 184 1.834035 1.285602 30.85 315506 unknown 99, 127 0.570919 0.400197 33.917 539799 ene al 0.976785 0.684696 34.083 234621 unsaturated acid? 0.424555 0.2976 34.233 290942 unknown 95, 123 0.52647 0.369039 35.133 243416 aldehyde? 0.44047 0.308756 35.617 733727 oxygenated 169, 226 1.327705 0.93068 36.8 314518 ene al 0.569131 0.398944 36.933 197544 ene al 0.357463 0.25057 37.95 733437 C7 alcohol 222 1.32718 0.930312 38.267 372053 acid 0.673243 0.471923 38.433 263295 aldehyde? 0.476442 0.333971 38.883 198355 alkene 0.35893 0.251599 39.55 244842 ene al? 0.44305 0.310565 39.667 469317 oxygenated 0.849246 0.595295 40.633 791154 aldehyde 1.431621 1.003522 40.8 358025 unsaturated alcohol? 0.647859 0.454129 42.433 4031365 C16 fatty acid 256 7.294898 5.113498 43.2 2228205 aldehyde 4.032016 2.826319 43.6 1397932 C17 fatty acid 270 2.529607 1.773177 43.75 389171 C17 fatty acid 270 0.704219 0.493636 44.467 1322757 C17 fatty acid 270 2.393576 1.677823 44.683 349965 alkene 0.633274 0.443906 46.233 409301 acid me ester 284 0.740645 0.519169 47.3 921726 C18 oxy acid me ester 1.667896 1.169144 47.717 369827 oxygenated unknown 0.669215 0.469099 48.883 374764 acid 298 0.678149 0.475361 49.483 2358985 alcohol? 4.268667 2.992204 50.983 1252071 unsat. Acid 292 2.265667 1.588163 51.35 681585 acid 312 1.233352 0.864542 51.9 13499889 alkane? 225, 255 24.42853 17.12364 53.067 1238206 alcohol? 2.240578 1.570576 53.833 410444 alcohol 0.742713 0.520619 56 9244792 alkane 16.72878 11.72636 60.4 1924170 sterol 351, 366 3.481854 2.440672 61.083 228826 sterol 349, 364 0.414069 0.290249 64.533 36058 sterol 301, 386 0.065248 0.045737 65.983 250884 sterol 269, 382 0.453983 0.318228 66.75 288328 sterol 287, 400 0.52174 0.365723 70.833 311785 sterol 287, 400 0.564186 0.395477 Chapter 6: Appendix 6.1 468

Appendix 6.1d Lepsiella reticularis R.t. intensity identity M+/ fragments %total 38.083 8579981 tyrindoleninone 255, 242 23.75596 41.669 4624625 C16 acid 256 12.80451 42.583 246145 brominated 227, 199 0.681518 43.117 1150508 aldehyde 3.185488 45.867 528294 ene al? 1.462723 46.433 2972534 C18 acid 284 8.230251 47.3 1097145 alkane 3.037738 47.4 495734 alcohol 1.372572 50.283 2284738 alcohol? 6.325905 50.4 1245921 alcohol? 3.449664 50.833 702556 alkane 1.945213 51.267 664651 aldehyde 1.840263 51.6 3514507 alkane 9.730848 53.633 4445433 alkane 12.30836 57.317 2697128 alkane 7.467717 65.783 867271 cholesterol 2.401271 Chapter 6: Appendix 6.1 469

Appendix 6.1e Morula marginalba 1 R. t. Area Identity M+/ fragments % total 38.067 1241844 tyrindoleneinone 240, 255 2.63304 41.667 7927904 fatty acid 16.80926 42.55 659863 bromoisatin 197, 225 1.399085 43.1 2406638 bromo/ aldehyde 241, 271 5.102713 44.8-45.6 4398392 tyriverdin 242, 257 9.325761 45.8 788954 ene al/ eneoic acid 264 1.672792 46.417 4901632 acid 284 10.39276 47.383 1563332 ether/ alcohol 3.31468 49.1 15671390 alcohol 33.22751 49.3 1030596 alcohol 2.185138 50.267 2295809 aldehyde 4.867726 51.25 1349174 aldehyde 2.860608 65.6 2553715 cholesterol 5.414555 66.75 374652 sterol 0.794362

Appendix 6.1e Morula marginalba 2 R. t. Area Identity M+/ fragments % total 28.65 266612 alkane 1.289976 35.483 563315 brominated 207, 209 2.725543 37.9 236875 aldehyde? 1.146096 38.033 962152 tyrindoleninone 4.655277 39.133 252904 brominated 257, 198 1.223651 41.633 3542491 C16 fatty acid 256 17.13999 42.55 812764 bromo indole dione 3.932478 43.083 1936495 aldehyde + brominated 241, 243 9.369538 45.8 385812 unsat. acid/ aldehyde 1.866713 46.417 1678562 acid 284 8.121555 47.383 703324 alkane 3.402963 49.05 2624530 amine 12.69853 49.367 521415 polyunsaturated 2.522815 50.25 683417 polyunsaturated 3.306645 50.817 336089 alkane 1.626133 51.233 431764 alkane 2.089047 52.717 1614431 amine 7.811263 55.567 1386523 amine 6.708553 59.517 745056 unknown 3.604879 65.433 983457 cholesterol 386 4.758359 Chapter 6: Appendix 6.1 470

Appendix 6.1f Trunculariopsis trunculus Fraction 1 R. t. Area Identity M+/ fragments % fraction % total 3.6-4.117 13.2 Di amino 107 5.178501 4.294079 5.4 4.8 Amine 167 1.883091 1.561483 5.55 8.3 C6 hydroxy ketone 3.256179 2.700065 10.08 0.6 Unknown 110, 125 0.235386 0.195185 10.3 1.1 Phenol 94 0.431542 0.35784 10.5 1.3 Nitrous acid ester? 100, 142 0.510004 0.422902 10.575 1 Acid 0.392311 0.325309 10.7 1.2 Propyl acetate 0.470773 0.390371 10.833 4.5 C8 yne 1.765398 1.463891 12.2 0.5 Yne ol? 108, 128 0.196155 0.162655 12.433 0.9 Unknown (pyrrolidinone) 148, 169 0.35308 0.292778 13.04 0.4 Thiosulphonate 108, 126 0.156924 0.130124 14.92 6.9 Unknown 56, 121 2.706944 2.244632 15.717 0.7 Acid 0.274617 0.227716 17.25 0.4 Amine? 87, 105 0.156924 0.130124 17.3 0.3 Unknown 110, 122 0.117693 0.097593 17.7 0.9 Unknown 107, 128 0.35308 0.292778 17.783 3.3 Unknown 77, 107 1.294625 1.07352 17.983 1 Oxy alcohol 0.392311 0.325309 18.517 1 Alkane 0.392311 0.325309 19.55 3.2 Unsat. Ketone 1.255394 1.040989 20.033 0.8 Di oic acid di me ester 0.313849 0.260247 20.617 2.3 Ene one 0.902315 0.748211 20.9 0.6 Alcohol 0.235386 0.195185 21.15 0.6 Acid 0.235386 0.195185 22.683 0.6 Yne 0.235386 0.195185 23.35 0.3 Benzoic acid amino me ester 133, 151 0.117693 0.097593 23.483 2.5 Ene one? 0.980777 0.813273 23.833 0.4 Unknown (sulfoxide?) 55, 106 0.156924 0.130124 24.1 2.1 Unknown 64, 146, 161 0.823852 0.683149 25.517 1.4 Alkane 0.549235 0.455433 27.86 1.4 Dibromo imidazole/pryazole 224, 226, 228 0.549235 0.455433 28 0.5 Ene? 0.196155 0.162655 28.468 3.3 Unknown 141, 181 1.294625 1.07352 28.783 5.5 Phenol 205, 220 2.157709 1.7892 29 0.5 Dibromo imidazole/pryazole 224, 226, 228 0.196155 0.162655 29.2 0.5 Unknown 161, 180 0.196155 0.162655 29.35 0.2 Bromo 414, 416 0.078462 0.065062 30.233 0.4 Unknown 196, 198 0.156924 0.130124 30.333 1.5 Methyl thioindolone 162, 177 0.588466 0.487964 30.433 0.5 Unknown 132, 174 0.196155 0.162655 30.65 0.4 Dibromo 316, 318, 320 0.156924 0.130124 30.8 1.1 Acid 0.431542 0.35784 30.87 0.4 Unknown 253, 262 0.156924 0.130124 31.417 19.9 Unknown 176, 177, 178 7.806983 6.47365 31.867 1.5 Alkane 0.588466 0.487964 32.317 0.7 Bromo 239, 241 0.274617 0.227716 32.6 0.6 Bromo 429, 431 0.235386 0.195185 33.55 6.4 Tribromo imidazole/pryazole 304 2.510789 2.081978

Chapter 6: Appendix 6.1 471

Appendix 6.1f Contd. Trunculariopsis trunculus R. t. Area Identity M+/ fragments % fraction % total 34.667 0.3 Indoledione 119, 135, 147 0.117693 0.097593 34.783 2.6 Alkane 1.020008 0.845804 34.9 0.4 Acid phenol ester? 95, 105 0.156924 0.130124 35.083 0.3 Benzene ethyl phenoxy 165, 183, 198 0.117693 0.097593 35.2 1 Ene one/al 0.392311 0.325309 36.2 0.2 Unknown 181, 196 0.078462 0.065062 36.55 1.7 Acid 0.666928 0.553025 37.983 1.2 Aldehyde 0.470773 0.390371 38.12 0.5 Tyrindoleninone 255, 257 0.196155 0.162655 38.233 15.6 Isopropyl myristate 6.120047 5.074821 38.783 10.8 Dibromo 294, 296, 298 4.236956 3.513338 39.183 0.8 Alcohol? 0.313849 0.260247 39.717 3.5 alcohol/ene 1.373087 1.138582 40.667 0.9 Aldehyde 0.35308 0.292778 41.217 1 alcohol or thiol 0.392311 0.325309 41.8 3.5 acid 1.373087 1.138582 42.55 0.3 bromoisatin 225, 227 0.117693 0.097593 43.217 2.1 Aldehyde/alcohol 0.823852 0.683149 43.317 0.9 acetic acid ester 0.35308 0.292778 44.233 1.1 acid 0.431542 0.35784 44.75 7.1 ol/ ene 2.785406 2.309694 49-45.8 0.3 brominated 257, 259 0.117693 0.097593 45.8 0.7 polyunsaturated 0.274617 0.227716 45.833 0.4 unknown 218, 219 0.156924 0.130124 45.983 1.4 ene al/ acid 0.549235 0.455433 46.083 0.7 unsat.aldehyde? 0.274617 0.227716 46.65 4.4 acid 1.726167 1.43136 46.883 4.5 amide 1.765398 1.463891 50.483 1.8 alcohol/aldehyde 0.706159 0.585556 50.6 0.9 alkene 0.35308 0.292778 50.817 4 amide 1.569243 1.301236 51.35 2.7 amide 1.059239 0.878334 51.417 0.6 C7 dioic acid di octyl ester 0.235386 0.195185 59.617 1.9 unknown 173, 217 0.74539 0.618087 60.683 1.4 sterol 368 0.549235 0.455433 61.083 0.5 sterol 364 0.196155 0.162655 61.433 0.6 sterol 370 0.235386 0.195185 61.733 0.6 sterol 379 0.235386 0.195185 63.55 2.1 sterol 384 0.823852 0.683149 63.867 2.1 sterol 385 0.823852 0.683149 64.683 40.5 cholesterol 386 15.88858 13.17502 64.95 2.4 sterol 430, 446 0.941546 0.780742 65.283 7.2 sterol 384 2.824637 2.342225 65.983 0.7 sterol 413, 430 0.274617 0.227716 66.283 0.4 sterol 398, 418 0.156924 0.130124 66.467 1.5 sterol 382, 401 0.588466 0.487964 68.25 1.2 sterol 397, 414 0.470773 0.390371 69.183 1.2 sterol 413, 424 0.470773 0.390371 Chapter 6: Appendix 6.1 472

Appendix 6.1f Trunculariopsis trunculus Fraction 2 R. t. Area Identity M+/ fragments % fraction % total 9.767 7.4 ethanol oxy bis 45, 47 14.09524 2.407287 10.4 1.5 unknown 60, 73 2.857143 0.487964 17.883 3.2 unknown 87, 100 6.095238 1.040989 19.517 6.2 unknown 83, 98 11.80952 2.016916 22.113 3.7 phthalic anhydride 104, 148 7.047619 1.203643 37.883 2 alkene 96, 111 3.809524 0.650618 41.7 12.1 unknown 40 23.04762 3.936239 43.083 7.5 aldehyde 110, 125 14.28571 2.439818 44.717 1.7 eneoic acid ester 110, 153 3.238095 0.553025 49.1 5.1 ether/ alcohol 73, 83, 95 9.714286 1.659076 59.483 2.1 unknown 217, 261 4 0.683149

Chapter 6: Appendix 6.1 473

Appendix 6.1h Conus paperliferus Fraction 1 R. t. Area Identity fragments % fraction % total 28.4 71617 unknown 6.519758 5.764264 37.933 15152 unsaturated oxygenated 164, 141 1.379384 1.219545 38.15 245226 isopropyl myristate 229, 211 22.32451 19.7376 39.633 16566 alcohol/alkene 1.50811 1.333354 40.6 16190 aldehyde 1.47388 1.30309 43.15 153986 aldehyde 14.01834 12.39393 59.55 81956 tri ene ol 368, 69 7.460984 6.596423 60.65 22941 unknown 360, 144 2.088467 1.846461 64.567 366188 cholesterol 33.33646 29.47351 64.733 22844 sterol 233, 207 2.079637 1.838653 64.867 45526 unknown 307, 293 4.144526 3.664268 65.217 40269 sterol 384 3.665947 3.241146

Fraction 2 R. t. Area Identity fragments % fraction % total 41.7 32232 fatty acid 227 22.388 2.594269 43.15 16375 ene one? 127, 111 11.3739 1.317981 54.167 12421 alkane 8.627492 0.999734 64.483 82942 sterol 387, 368 57.61061 6.675783

Chapter 6: Appendix 6.1 474

Appendix 6.1g Mitra carbonaria R. t. Area Identity fragments/M+ % total 34.683 4.4 dione? 114 0.996151 37.85 4.8 aldehdye/alcohol 1.08671 38.083 36.4 propyl tetranoate 229 8.240887 39.55 3.9 alcohol 0.882952 40.517 7.2 alcohol 1.630066 42.133 5.6 oxygenated 1.267829 43.067 59.7 aldehyde/alcohol 13.51596 45.733 12.2 alkene? 2.762056 48.033 12.7 aldehyde/alcohol 2.875255 51.25 4.7 hexanedioic acid ethylhexylester 1.064071 51.7 5.3 ene one 1.199909 60.917 11.9 sterol 364 2.694136 61.333 3.7 sterol 364 0.837673 63.65 6.7 sterol 310 1.516867 64.4 210.1 cholesterol 386 47.56622 65.05 21.4 cholestadieneol 384 4.844917 65.433 3 sterol 369 0.679194 66.067 1.6 sterol 314 0.362237 66.2 3.5 sterol 213 0.792393 67.967 11.6 sterol 330 2.626217 68.85 5.7 sterol 412 1.290469 68.9 5.6 sterol 412 1.267829

Chapter 6: Appendix 6.1 475

Appendix 6.1i Aplysia sp. Freeze dried eggs Fraction 1 R. t. intensity identity M+/ fragments % fraction % total 4.117 4965504 urea 44, 60 3.764355 3.686109 5.517 295204 urea (ethanediol diformate) 60, 72 0.223795 0.219143 6.9 418018 chlorinated 0.3169 0.310313 9.7 817024 trichloro propanone 0.619387 0.606512 10.117 686893 chlorinated 0.520734 0.509911 10.75 778351 chlorinated 0.590069 0.577804 11.633 622118 trichloro propanone 0.471628 0.461825 12.05 158436 chlorinated 0.120111 0.117614 12.167 235133 dichloro ketone 0.178255 0.174549 12.417 204639 chlorinated 0.155137 0.151912 12.667 141751 chlorinated 0.107462 0.105228 12.933 343522 unknown 43, 59 0.260424 0.255011 13.533 1952401 alkane 1.480118 1.449352 13.767 153236 chlorinated 337, 123, 101 0.116168 0.113754 13.933 332092 ketone 0.251759 0.246526 14.267 1991403 trichloro ene 1.509685 1.478305 14.95 597040 chlorinated 0.452617 0.443209 15.567 428677 unknown 107, 93, 90 0.324981 0.318226 16.6 317147 chlorinated 0.24043 0.235432 16.967 144515 thio chloro? 0.109557 0.10728 17.283 685961 dichloro propanal 0.520028 0.509219 17.683 128952 dichloro 0.097759 0.095727 17.967 248290 alcohol 59, 41 0.188229 0.184316 18.433 474423 chlorinated 0.359661 0.352185 18.817 63679 chlorinated 0.048275 0.047272 19.15 2138198 chlorinated 1.620971 1.587277 19.483 4084839 chlorinated 3.096722 3.032353 19.583 248278 tetrachloro 0.18822 0.184308 20.1 196590 chlorinated 0.149035 0.145937 20.233 168626 dichloro ene 0.127836 0.125178 20.65 116133 dichloro ene 0.088041 0.086211 21.25 494211 chlorinated 0.374662 0.366874 21.8 8776675 alcohol 6.653609 6.515307 21.883 1586843 chlorinated 1.202988 1.177982 22.233 220450 dichloro ene 0.167123 0.16365 22.3 184510 chlorinated 0.139877 0.13697 22.683 363051 unsaturated (yne) 0.275229 0.269509 23.3 545976 oxygenated 0.413905 0.405302 24.317 351042 tetrachloro 0.266125 0.260594 25.267 750420 unknown 0.568894 0.557069 26 969871 unknown 93, 71, 57 0.73526 0.719977 26.1 513757 polychlorinated 110, 62 0.38948 0.381384 26.8 427776 chloro alcohol 59, 43 0.324298 0.317557 34.067 244630 alcohol 236 0.185454 0.181599 36.133 776864 alkane 0.588942 0.5767 36.9 819423 acid 228 0.621206 0.608293 37.167 644723 aldehyde 0.488765 0.478606 39 589680 acid 228 0.447037 0.437745 Chapter 6: Appendix 6.1 476

Appendix 6.1i Aplysia sp. Freeze dried eggs Fraction 1 continued. R. t. intensity identity M+/ fragments % fraction % total 39.65 529116 acid 0.401124 0.392786 39.85 236184 aldehyde 0.179051 0.17533 40.15 282838 aldehyde 0.21442 0.209963 40.633 304706 ketone 0.230998 0.226196 40.767 1083138 aldehyde 0.821128 0.80406 41.45 349156 me ester 270 0.264696 0.259194 41 744015 eneoic acid 236 0.564039 0.552315 41.9 438704 eneoic acid 0.332582 0.325669 42.25 15189481 acid 256 12.55985 11.32535 43.6 654802 alkane 0.496406 0.486088 43.683 287186 acid 0.217716 0.213191 43.867 1543295 acetic acid ester? 1.169974 1.145655 44.05 602372 acid 270 0.456659 0.447167 44.483 4670281 alcohol 3.540546 3.466952 44.567 6102527 aldehyde 250 4.626334 4.530171 44.95 2037832 acid 1.544883 1.512771 45.017 631713 alcohol 0.478902 0.468948 45.933 896850 yne ol 0.679903 0.665771 46.067 825735 ene yne 0.625991 0.612979 46.867 4805865 yne ol 3.643333 3.567602 47.467 10741244 eneoic acid 8.142951 7.973692 47.983 11131060 acid 8.438471 8.263069 48.133 1083723 eneoic acid 2.624466 2.511485 48.967 686106 eneoic acid 0.520138 0.509326 50.283 1769619 ene yne 1.341551 1.313665 51.017 4789498 ene yne 3.630925 3.555452 51.4 8825064 eneoic acid 6.657887 6.581855 51.617 1236499 amide 0.937392 0.917907 54.1 1175008 ene yne 0.890775 0.872259 54.233 1264999 ene yne 0.958997 0.939064 64.667 1334036 cholesterol 386 1.011334 0.990313 65.283 676352 sterol 384 0.512743 0.502085

Chapter 6: Appendix 6.1 477

Appendix 6.1i Aplysia sp. Freeze dried eggs Fraction 2 continued. retention time intensity identity fragments % fraction % total 4.433 59303 amine 2.117926 0.044023 8.1 69763 chloroacetic acid 2.49149 0.051788 10.683 65169 tetrahydrofuran dimethyl 103, 85 2.327422 0.048378 17.95 138495 piperidinone 99, 42 4.94616 0.102811 20.517 90582 benzene acetic acid 136, 91 3.235013 0.067243 30.75 37671 acid 1.345368 0.027965 36.5 56631 acid 2.022499 0.04204 41.217 23520 eneoic acid me ester 0.839985 0.01746 41.883 657240 acid 256 23.47243 0.487898 43.15 239202 aldehyde/ alcohol 8.542773 0.17757 43.483 85301 acetic acid ester 3.046409 0.063323 44.183 53036 acid 1.894108 0.039371 44.383 95561 aldehyde/ acetic acid ester 3.412831 0.070939 45.783 59558 triene 2.127033 0.044212 45.817 60507 yne 2.160925 0.044917 45.933 131936 eneoic acid 4.711914 0.097942 46.083 140255 eneoic acid 5.009016 0.104117 46.583 312987 acid 284 11.1779 0.232344 49.817 40769 yne ol 1.456009 0.030265 49.933 992514 unsaturated oxy 292 6.904011 0.148677 54.083 50321 polyunsaturated oxy 1.797146 0.037355 60.65 42466 sterol 368 1.516615 0.031524 61.35 34382 sterol 1.227906 0.025523 61.7 60027 sterol 366 2.143782 0.044561

Chapter 6: Appendix 6.1 478

Appendix 6.1j Aplysia juliana Frozen eggs Fraction 1 R. t. Area Identity M+/ fragments % fraction % total 4.483 12.1 hexenal 0.677753 0.346189 10 5.7 ? acetyl? 110 0.319272 0.163081 10.783 5.3 heptadienal 110 0.296867 0.151637 11.317 3.5 heptadienal 110 0.196044 0.100137 14.717 8.8 C11 alkane 156 0.492912 0.251774 15.617 2.8 C8 acid me ester 0.156836 0.08011 17.9 10.6 oxy ethoxy ethanol 0.593734 0.303273 18.467 44.6 C12 alkane 170 2.498166 1.276036 18.917 8.2 alkane 0.459304 0.234607 21.033 9.2 C13 alkane 184 0.515317 0.263218 21.633 4 unsaturated 0.224051 0.114443 21.817 7.9 yne 152 0.4425 0.226024 22.05 31 C13? alkane 184 1.736393 0.886931 22.633 11.7 decadienal 152 0.655348 0.334745 25.467 6.21 C14 alkane 198 0.347839 0.177672 25.783 1.8 unknown 127 0.100823 0.051499 28.4 11.2 unknown 141 0.627342 0.320439 28.717 4.9 alkane 0.274462 0.140192 29.117 7.2 unknown (diene?) 124, 137, 180 0.403291 0.205997 29.433 5 C10 fatty acid me ester 186 0.280063 0.143053 30.85 6.7 unknown 127 0.375285 0.191691 31.133 2.4 unsaturated alcohol 174 0.13443 0.068666 31.233 3.3 poly unsaturated 0.184842 0.094415 35.4 39.6 C14 acid me ester 242 2.218102 1.132982 36.650-37.067 8.9 C14 acid 228 0.498513 0.254635 37.917 3.2 alkane 0.179241 0.091554 38.333 8.4 oxygenated 0.470507 0.24033 39.517 9.5 C15 acid 242 0.53212 0.271801 39.6 10.6 C16 acid 256 0.593734 0.303273 39.817 4 C17 acid 270 0.224051 0.114443 40.233 37.3 C16 eneoic acid me ester 268 2.089273 1.067178 40.967 155.9 C16 acid me ester 270 8.732377 4.460403 41.717 10.9 acid 0.610538 0.311856 42.05 17.6 C18 acid 284 0.985823 0.503548 42.65 87.2 C18 acid 284 4.884306 2.49485 43.00-43.967 139.6 acid 7.81937 3.994049 44.25 34.7 acid 1.94364 0.99279 44.833 41.4 acid 270 2.318925 1.184482 45.25 100.5 acid me ester? 5.629274 2.875372 45.45 100.6 C18 unsaturated me ester 296 5.634876 2.878233 45.9 111.6 C18 me ester 298 6.251015 3.19295 47.050-47.800 98.5 alcohol/aldehyde 280 5.517249 2.818151 48.2 113.1 C18 acid 284 6.335034 3.235866 48.667 7.6 polyunsaturated acid 0.425696 0.217441 48.8 14.3 alcohol? 284 0.800981 0.409133 49.15 15.5 C12 trieneoic acid me ester 320 0.868197 0.443465 49.3 32.6 C21 dieneoic acid me ester 322 1.826013 0.932708 49.6 14.5 C21 diene me ester 322 0.812184 0.414855 49.8 48.6 C21 eneoic acid me ester 324 2.722216 1.390478 Chapter 6: Appendix 6.1 479

Appendix 6.1j Aplysia juliana Frozen eggs Fraction 1 continued. R. t. Area Identity M+/ fragments % fraction % total 49.917 18.8 C21 eneoic acid me ester 324 1.053038 0.537881 51.35 90.7 C21 trieneoic acid 306 5.08035 2.594987 51.65 65.6 C21dieneoic acid 308 3.674432 1.87686 51.733 66.6 C21 dieneoic acid 308 3.730445 1.90547 52.95 4.2 trieneoic acid me ester 0.235253 0.120165 53.083 5.2 trienoic acid 0.291266 0.148775 53.4 4.8 C22? dieneoic acid me ester 336 0.268861 0.137331 54.783 5.5 unknown 103 0.30807 0.157359 55.183 9.3 unknown 103 0.520918 0.266079 55.7 4 unknown 103 0.224051 0.114443 56.1 3.8 unknown 103 0.212848 0.108721 61.083 1.9 sterol 364 0.106424 0.05436 64.55 18.7 sterol 386 1.047437 0.535019 65.133 5.9 sterol 369 0.330475 0.168803

Chapter 6: Appendix 6.1 480

Appendix 6.1j Aplysia juliana Frozen eggs Fraction 2 R. t. Area Identity M+/ fragments % fraction % total 4.45 88 hexanal 5.1465 2.517739 9.983 15.7 amine? 68, 110 0.918182 0.449187 10.483 11.9 C9 dieneal? 138 0.695947 0.340467 10.767 6.7 dienal? 0.391836 0.191691 13.3 26.7 ketone 1.561495 0.763905 14.45 16.9 enal 0.988362 0.48352 21.783 11.4 alkyne (or dienal) 0.666706 0.326162 22.6 23.4 unknown 152 1.368501 0.66949 23.867 17.2 C8 ketone 128 1.005907 0.492103 24.867 7.5 acid? 0.438622 0.21458 26.467 5.3 acid 0.30996 0.151637 28.083 7.3 acid 0.426926 0.208858 30.717 8.8 unknown 127 0.51465 0.251774 34 8.9 alcohol 0.520498 0.254635 34.133 15.6 unknown 123, 193 0.912334 0.446326 36.35 13.5 ene one 135, 140, 178 0.78952 0.386244 36.433 12.9 C14 acid 228 0.75443 0.369078 37.9 15.5 C12 aldehyde? 0.906486 0.443465 39.483 10.7 enal/ol 0.625768 0.306134 39.567 9.1 alcohol 0.532195 0.260357 39.783 8.1 alcohol/aldehyde 0.473712 0.231746 40.55 20 aldehyde/alcohol 1.169659 0.572213 41.367 53.6 C11 eneal 168 3.134686 1.533532 41.867 76 C16 acid 256 4.444704 2.174411 43.133 192.4 aldehyde? 11.25212 5.504692 44.333 132.7 ene ol 7.760688 3.796635 44.6 20.7 ene al 1.210597 0.592241 45.9 14.5 unknown amide? 0.848003 0.414855 51.583 433.1 enyl oxy ether or alcohol 25.32897 12.39128 53.033 26.6 alcohol 1.555647 0.761044 54.483 33.7 unknown 103 1.970875 0.964179 54.9 34 unknown 103 1.98842 0.972763 55.483 14.7 unknown 103 0.859699 0.420577 55.7 160.8 unknown enyl oxy 9.404059 4.600595 55.883 16 unknown 103 0.935727 0.457771 56.667 22.1 unknown sterol ? 1.292473 0.632296 59.55 36.2 sterol? 261, 363 2.117083 1.035706 60.983 37.3 sterol? 251, 364 2.181414 1.067178 64.467 44.4 sterol? 364, 382 2.596643 1.270314 Chapter 6: Appendix 6.1 481

Appendix 6.1j Aplysia juliana derivatised R. t. Area Identity M+/ fragments % fraction 35.4 9740309 C14 me ester 242 2.173167 36.717 2188014 me ester 0.488169 38.15 5416886 C15 me ester 256 1.208565 39.517 1085134 polyunsaturated 0.242105 39.85 762855 unsaturated 0.170201 40.017 2772437 C16 eneoic acid me ester 268 0.61856 40.183 6437220 C16 eneoic acid me ester 268 1.436213 40.8 40673100 C16 me ester 270 9.074604 41.4 6356027 phthalate 1.418098 41.717 10511337 C16 acid 256 2.345192 42 3959267 C17 me ester 284 0.883355 42.55 18581918 C17 me ester 284 4.145825 42.633 2550621 C17 eneoic acid 282 0.569071 42.983 8122537 dimethoxy/ ether 250 1.812225 43.15 10835134 aldehyde 250 2.417434 43.267 11607195 C17 me ester 284 2.589689 43.7 2334522 dimethoxy/ ether 75 0.520857 43.867 4841241 dimethoxy/ ether 71 1.080133 44.167 3256955 dimethoxy/ ether 0.726662 44.367 8985974 aldehyde 2.004867 44.5 10740478 C18 tetra enoic me ester 290 2.396316 44.817 13417797 C18 ynoic acid me ester 294 2.993654 44.95 15939908 C18 trieneoic me ester 292 3.556364 45.133 13870437 C18 eneoic me ester 296 3.094643 45.667 21401991 C18 me ester 298 4.775013 45.867 2033312 unsaturated oxygenated 0.453654 45.983 2817483 C18 eneoic acid (oleic) 282 0.628611 46.067 1202660 C18 eneoic acid (oleic) 282 0.268326 46.483 7560839 C18 acid 284 1.686904 46.717 20686473 unknown 283, 175 4.615373 47.25 1234690 acid me ester 312 0.275473 47.383 908760 eneoic acid me ester 0.202754 47.45 1364763 eneoic acid me ester 310 0.304493 47.817 17052559 unknown 297, 75 3.804608 48.317 5999158 unknown 298, 75 1.338476 48.467 15018559 C20 tetraeneoic me ester 318 3.350801 48.583 13554797 C20 pentaenoic me ester 316 3.02422 48.8 8854153 C20 trieneoic me ester 320 1.975456 48.95 11706197 C20 trieneoic me ester 320 2.611778 49.033 14730740 C20 tetraeneoic me ester 318 3.286586 49.367 9588381 C20 dieneoic me ester 322 2.13927 49.5 13070631 C20 eneoic acid me ester 324 2.916198 49.65 8816305 C20 eneoic acid me ester 324 1.967012 49.9 965639 unsaturated alcohol 304 0.215444 50.1 1607696 C20 me ester 326 0.358694 50.3 1062331 eneoic acid me ester 0.237017 50.5 880484 eneoic acid me ester 0.196445 51.133 750312 dieneoic acid me ester 0.167403 51.217 1586084 dieneoic acid me ester 0.353872 51.767 967629 eneoic acid me ester 0.215888 Chapter 6: Appendix 6.1 482

Appendix 6.1j Aplysia juliana derivatised continued. R. t. Area Identity M+/ fragments % fraction 52.3 1849403 unknown 294, 141 0.412622 52.5 894903 polyunsaturated 0.199662 52.7 12488670 C22 tetraenoic me ester 346 2.786356 52.817 11584586 C22 penteneoic me ester 344 2.584645 53.05 1792464 C22 trieneoic acid me ester? 0.399918 53.15 10302354 C22 dieneoic acid me ester 350 2.298565 53.217 2923984 C22 dieneoic acid me ester 0.652372 53.617 901973 eneoic acid me ester 0.20124 65.317 3767178 cholesterol 386 0.840498 Chapter 6: Appendix 6.1 483

Appendix 6.1k Aplysia parvula derivatised R. t. intensity Identity M+/ fragments % total 11.782 7932023 unknown 112, 98 2.148606 29.5 882968 me ester 0.239176 35.417 4011452 me ester 1.086612 36.767 615853 me ester 0.166821 38.067 475409 di phenyl 236, 221 0.128778 38.2 2059125 me ester 0.55777 40.2 2848267 C16 ene oic me ester 0.771531 40.817 19934174 C16 me ester 270 5.399717 41.733 714406 C 16 acid 242 0.193516 42.05 1718268 me ester 0.46544 42.4 925991 me ester 284 0.25083 42.583 11854349 me ester 284 3.211075 42.667 1615654 alcohol 0.437644 43.167 1908326 C16 vinyl ether 268, 253 0.516922 43.3 5825392 me ester 1.577967 43.883 9039384 t-butyl dimethyl benzyl phenol 324, 309 2.448565 44.433 1496571 phthalate 0.405387 44.55 1715036 methyl eicosatetraenoate 0.464564 44.85 5820691 di eneoic me ester 1.576693 45.017 10500198 C18 eneoic me ester 2.844266 45.167 11165374 C18 eneoic me ester 296 3.024447 45.7 13328939 C18 me ester 298 3.610508 46.767 11666474 di methoxy 3.160184 47.367 12019326 C22 alkane 310 3.255763 47.85 5823409 di methoxy 1.57743 48.5 10661184 tetraeneoic me ester 2.887873 48.617 12647629 di yne oic me ester 3.425956 48.5 7355087 trieneoic me ester 320 1.992327 49.067 7514032 trieneoic me ester 2.035381 49.4 2771908 dieneoic me ester 322 0.750847 49.567 13007503 eneoic me ester 324 3.523438 49.667 2310129 eneoic me ester 0.625762 50.117 1239669 methyl dehydroabietate 239, 299, 314 0.335798 50.339 1049583 alkane 0.284308 51.683 13340496 alkane 3.613638 52.167 567781 dehydroabietic acid 239, 285, 300 0.153799 52.733 7223360 trieneoic me ester 394 1.956645 52.85 5064409 trieneoic me ester 1.371834 53.167 3264389 dieneoic me ester 0.884249 53.3 5168900 dimethyl benzyl phenol 330, 315, 237 1.400138 53.483 15422609 dimethyl benzyl t-butyl phenol 386, 371, 293 4.177636 53.717 13490778 alkane 3.654346 54.967 2759793 alkane 0.747565 55.7 13373971 alkane 3.622706 56.883 3257006 alkane 0.882249 57.583 12378432 alkane 3.353037 58.75 3072431 alkane 0.832252 58.933 1621507 alkane 0.43923 59.45 12240559 alkane 3.31569 60.567 2648117 alkane 0.717315 Chapter 6: Appendix 6.1 484

Appendix 6.1k Aplysia parvula derivatised R. t. intensity Identity M+/ fragments % total 61.233 11762687 alkane 3.186246 63.016 10798532 alkane 2.925078 65.067 10162197 alkane 2.752709 65.717 2150872 cholesterol 386 0.582623 66.65 941879 sterol 384 0.255134 67.567 8043013 alkane 2.17867 70.567 5963242 alkane 1.615307

Chapter 6: Appendix 6.1 485

Appendix 6.1l Aplysia sydneyensis Derivatised R. t. intensity Identity M+/ fragments % total 10.017 2438540 dimethoxy 75, 115 2.433531 31.583 1276581 dimethoxy 1.273959 35.333 2062374 C14 me ester 242 2.058137 36.633 1040781 C14 me ester 1.038643 38.083 1600863 C15 me ester 256 1.597574 40.117 1332858 me ester 1.33012 40.7 17137868 me ester 270 17.10266 41.633 564104 C16 acid 0.562945 41.95 890660 me ester 0.88883 42.283 514293 me ester 0.513236 42.467 6043172 me ester 284 6.030758 42.917 901099 dimethoxy 71, 82 0.899248 43.083 1268482 aldehyde/ ene ol 1.265876 43.217 1611395 me ester 284 1.608085 44.333 1157994 dimethoxy 75 1.155615 44.433 1391942 trieneoic me ester 268 1.389083 44.733 3821790 dieneoic me ester 294 3.813939 44.867 8860544 trieneoic me ester 292 8.842342 45.05 7270060 C18 eneoic me ester 298 7.255125 45.583 12517340 C18 me ester 12.49163 48.367 2353176 tetraeneoic me ester 394 2.348342 48.517 7466137 trieneoic me ester 7.450799 48.733 680930 trieneoic me ester 0.679531 48.833 2362230 trieneoic me ester 2.357377 48.983 2836138 trieneoic me ester 2.830312 49.3 2010278 C21 dieneoic me ester 322 2.006148 49.45 1466290 eneoic me ester 1.463278 49.583 1983023 eneoic me ester 1.978949 52.633 1682795 trieneoic me ester 1.679338 52.75 1536582 trieneoic me ester 1.533425 53.067 1246861 dieneoic me ester 1.2443 53.183 431968 dieneoic me ester 0.431081 65.5 446704 cholesterol 386 0.445786

Chapter 6: Appendix 6.1 486

Appendix 6.1m Stylocheilus longicauda Fresh eggs Fraction 1 R. t. Area Identity M+/ fragments % fraction % total 10.033 66832 pyrroline 110, 68 0.149722 0.09809 10.8 206611 heptadienal 110 0.462865 0.303246 11.35 72735 diene 0.162946 0.106754 12.167 76880 dihydropyranone 98, 68 0.172232 0.112838 13.367 136567 ester 259, 115 0.305947 0.200442 19.017 193070 unknown 112, 110 0.432529 0.283372 19.383 219527 di/tri chloro alkane 110, 65 0.4918 0.322203 21.05 94102 unknown 105, 93 0.210814 0.138115 21.633 1753098 chloro 117, 87 3.927415 2.57305 21.717 352560 unknown 117, 91 0.78983 0.517458 21.85 268371 alkyne 124, 95 0.601224 0.393892 22.1 127027 unknown 153, 141 0.284575 0.18644 22.517 85642 unknwon 164, 152 0.191861 0.125698 22.65 146067 alkyne 175, 153 0.32723 0.214385 23.233 115860 oxygenated 106, 93 0.259558 0.17005 24.867 57816 unknown 129, 99 0.129524 0.084857 25.2 224057 unknown 83, 76 0.501948 0.328852 25.833 117097 unknown 127 0.262329 0.171865 25.933 278552 di chloro 93, 83 0.624032 0.408835 26.067 133546 poly chlorinated 173, 157 0.299179 0.196008 26.75 111239 glycol 92, 83 0.249205 0.163267 27.917 142428 unsaturated ketone 177, 91 0.319077 0.209044 28.033 84579 unknown 158, 123 0.18948 0.124138 28.417 122982 unknown 141 0.275513 0.180503 28.717 286946 alkane 0.642837 0.421155 30.783 57411 acid 0.128616 0.084263 31.15 139103 yne 91, 81 0.311628 0.204164 31.283 369426 ene yne 137, 107 0.827614 0.542212 32.333 98372 di yne 95, 91 0.22038 0.144382 34.733 112327 ketone 209, 192 0.251643 0.164864 35.133 98871 unsaturated alcohol 124, 109 0.221498 0.145115 35.383 226794 me ester 199 0.50808 0.332869 35.483 109919 ester 0.246248 0.16133 36.933 689600 acid 1.544891 1.012137 37.95 193368 aldehyde 0.433197 0.283809 38.167 1069893 ester 2.39685 1.570299 38.25 582819 alcohol 1.305672 0.855413 38.433 235194 acid 0.526898 0.345198 38.6 102556 ether/alcohol 0.229753 0.150523 39.333 221958 acid 0.497246 0.325771 39.433 100562 di yne 0.225286 0.147596 39.533 68362 ene one 0.153149 0.100336 39.7 108479 ene 0.243022 0.159216 40.217 734718 C16 eneoic acid me ester 1.645967 1.078357 40.5 84463 polyunsaturated 0.18922 0.123968 40.617 277363 aldehyde 0.621368 0.40709 40.783 706411 acid me ester 1.582552 1.036811 41.933 1577516 C18 eneoic acid me ester 3.534063 2.315345 42.783 3858622 acid 8.644359 5.663361 Chapter 6: Appendix 6.1 487

Appendix 6.1m Stylocheilus longicauda Fraction 1 continued. R. t. Area Identity M+/ fragments % fraction % total 43.2 509979 aldehyde 1.142491 0.748504 43.717 716725 acid 1.605658 1.051949 43.917 656455 C17 acid 1.470637 0.963489 44.017 231794 alcohol 0.519281 0.340208 44.5 564855 acid 1.265428 0.829047 45.883 979200 unsaturated alcohol 2.193673 1.437187 46.667 2029567 eneoic acid 4.54678 2.978828 46.8 2315189 eneoic acid 5.186651 3.39804 47.167 2137364 acid 4.788274 3.137043 48.15 161236 aldehyde 0.361212 0.236649 48.917 221007 unknown 0.495116 0.324375 50.133 1910131 polyunsaturated 4.279211 2.80353 50.233 682515 C21 trieneoic acid me ester 1.529019 1.001738 50.383 706844 unsaturated 109, 96 1.583522 1.037446 50.567 588029 unsaturated 1.317344 0.86306 50.767 1033841 eneoic acid 2.316084 1.517385 50.917 1138158 unknown 292 2.549782 1.670493 53.867 407014 polyunsaturated me ester 133, 119 0.911822 0.597381 54 486108 polyunsaturated 108, 93 1.089014 0.713468 58.917 139953 acid 0.313533 0.205411 59.667 6727318 trieneol 341 15.07102 9.873792 61.117 261246 sterol 364 0.585262 0.383435 61.383 71385 unknown 381, 364 0.159922 0.104773 61.717 158224 unknown 367, 350 0.354465 0.232228 64.633 1335634 cholesterol 386 2.992182 1.960331 65.35 707358 sterol 384 1.584674 1.038201 66 107826 sterol 396 0.241559 0.158258 66.45 112339 sterol 400 0.25167 0.164882 67 85031 sterol 394 0.190492 0.124801 68.283 126985 sterol 400 0.284481 0.186378 68.6 29850 sterol 395 0.066872 0.043811 Chapter 6: Appendix 6.1 488

Appendix 6.1m Stylocheilus longicauda Fraction 2 R. t. Area Identity M+/ fragments % fraction % total 4.483 160703 hexanal 97. 85 0.68397 0.235866 10.033 124597 pyrazole or furandione 0.530299 0.182873 11.317 92069 benzyl chloride 0.391856 0.135131 12.183 134876 dihydropyranone (closest) 0.574047 0.19796 13.95 116846 furane 0.49731 0.171497 14.417 147944 bromo methyl benzene 0.629666 0.21714 17.633 9382 unknown 0.039931 0.01377 17.933 116956 unknown oxygenated 0.497778 0.171658 19.483 48485 unknown 0.206358 0.071162 19.6 211134 hexeneone 0.89861 0.309885 20.55 133688 benzeneacetic acid 136, 91 0.568991 0.196216 20.683 228777 alkane 0.973701 0.33578 21.667 75977 unsaturated 121, 113 0.323367 0.111513 21.817 71148 yne 218, 152 0.302814 0.104425 22.35 69629 methyl propyl dioxepane 136, 123 0.296349 0.102196 22.5 66733 unknown 152, 137 0.284023 0.097945 22.633 76436 yne 81 0.32532 0.112186 23.2 57395 unknown 172, 127 0.24428 0.08424 24.767 85416 unknown 128, 82 0.36354 0.125366 24.833 123400 unknown 127 0.525204 0.181116 28.133 52100 unknown 151, 113 0.221743 0.076468 30.4 86536 unknown 174, 132 0.368307 0.12701 30.833 124607 C12 acid 0.530341 0.182888 35.483 76051 alkane 0.323682 0.111621 35.517 84288 acid 0.358739 0.123711 36.883 976786 acid 4.157312 1.433644 38.067 299995 oxygenated 1.276813 0.440307 38.317 149182 acid 0.634935 0.218957 38.467 104756 eneal 0.445853 0.153752 39.267 257581 acid 1.096294 0.378056 39.55 98364 aldehyde 0.418648 0.14437 40.833 108954 ene yne 0.463721 0.159914 41.617 1452293 eneoic acid 254 6.181123 2.131554 42.5 3494160 acid 256 14.87154 5.128434 43.2 429560 aldehyde 1.828256 0.630472 43.767 936066 acid 3.984003 1.373879 43.95 743812 C17 acid 270 3.165748 1.091705 44.05 106572 eneoic acid 223, 208 0.453582 0.156417 44.533 770166 acid 3.277913 1.130385 44.683 91894 ene al 281, 270 0.391111 0.134874 45.817 480362 ene yne 102, 93 2.044475 0.705035 46.55 2164791 enoic acid 264 9.213595 3.177298 47.1 2495222 acid 284 10.61995 3.662277 49.2 252429 dioxalone/alcohol 73 1.074366 0.370494 49.933 1295566 polyunsaturated 166, 147 5.514075 1.901523 50.05 349241 polyunsaturated 1.486409 0.512587 50.167 438331 yne ol/ ene yne 1.865586 0.643345 50.417 303226 ene ol 109, 95 1.290564 0.44505 50.617 645293 eneoic acid 2.74644 0.947107 50.767 916375 enoic acid 292 3.900196 1.344978 Chapter 6: Appendix 6.1 489

Appendix 6.1m Stylocheilus longicauda Fraction 2 continued. R. t. Area Identity M+/ fragments % fraction % total 51.067 215936 acid 0.919048 0.316933 53.583 122053 ene yne 0.519471 0.179139 53.717 229504 C21 trieneoic acid me ester 0.976795 0.336847 53.833 194925 unsaturated 145, 131 0.829623 0.286095 54.567 242931 hydroxy ester 103 1.033942 0.356554 55 243032 hydroxy ester 103 1.034372 0.356702 58.867 146037 unknown 368 0.62155 0.214341 64.4 165049 sterol 364, 275 0.702467 0.242245 Chapter 6: Appendix 6.1 490

Appendix 6.1n Philine angasi Fraction 1 R. t. Area Identity M+/ fragments % fraction 1 % total 23.35 0.5 unsaturated ? 110 0.447628 0.163666 25.733 1.9 unknown 167 1.700985 0.621931 28.317 3.6 unknown 181 3.222919 1.178396 28.633 0.2 unknown 222 0.179051 0.065466 29.033 0.2 unknown 217 0.179051 0.065466 29.767 0.3 unknown 268 0.268577 0.0982 34.483 0.4 phenyl ketone? 204 0.358102 0.130933 35.05 0.4 aldehyde 0.358102 0.130933 35.333 5.4 C14 acid me ester 242 4.834378 1.767594 36.833 0.4 unsaturated alcohol? 0.358102 0.130933 37.25 0.5 ethyl ester 0.447628 0.163666 37.867 1.3 aldehyde 1.163832 0.425532 38.067 1.7 methyl ester 1.521934 0.556465 38.683 0.5 unknown 290 0.447628 0.163666 39.55 1.9 alcohol 1.700985 0.621931 39.733 1.2 methyl ester 1.074306 0.392799 40.1 0.9 alkene 0.80573 0.294599 40.517 1.9 aldehyde 1.700985 0.621931 40.7 16.8 C16 acid me ester 270 15.04029 5.499182 41.833 0.3 unsaturated aldehyde 0.268577 0.0982 42.117 0.7 unsaturated ketone 0.626679 0.229133 42.283 1.3 C17 acid me ester 284 1.163832 0.425532 42.417 1 ethyl ester 0.895255 0.327332 42.467 1.1 C17 acid me ester 284 0.984781 0.360065 43.067 6.2 aldehyde/ alcohol 5.550582 2.02946 43.183 1.5 C18 acid me ester 298 1.342883 0.490998 44.917 0.9 eneoic acid methyl ester 0.80573 0.294599 45.05 1.7 C18 enoic acid methyl ester 296 1.521934 0.556465 45.567 5 C18 acid me ester 298 4.476276 1.636661 47.1 0.3 C18 acid ethyl ester 312 0.268577 0.0982 47.3 1.7 aldehyde? 1.521934 0.556465 48.383 0.9 poly unsaturated 0.80573 0.294599 48.5 1.6 poly unsaturated 1.432408 0.523732 48.717 0.5 unknown 281 0.447628 0.163666 48.933 0.3 diene 0.268577 0.0982 49.367 0.4 unknown 0.358102 0.130933 49.567 0.9 eneoic acid me ester 0.80573 0.294599 49.867 0.4 unknown 0.358102 0.130933 51.267 1.2 C22? dioic acid ester 1.074306 0.392799 52.417 0.2 unsaturated methyl ester 0.179051 0.065466 52.733 0.6 unsaturated aldehyde or alcohol 0.537153 0.196399 63.667 1.6 cholestadienol 384 1.432408 0.523732 64.433 26.9 cholesterol 386 24.08236 8.805237 64.533 2 cholestanol 388 1.79051 0.654664 65.033 6.7 methylcholestadienol 398 5.998209 2.193126 66.05 0.7 methylcholestenol 400 0.626679 0.229133 66.233 1.1 sterol 401 0.984781 0.360065 66.75 1.2 sterol 412 1.074306 0.392799 Chapter 6: Appendix 6.1 491

Appendix 6.1n Philine angasi continued. Fraction 2 R. t. Area Identity M+/ fragments % fraction 1 % total 68 2.5 sterol 414 2.238138 0.818331 68.333 0.3 sterol 411 0.268577 0.0982 4.45 0.8 hexanal 100 0.412797 0.261866 7.383 0.9 C7 aldehyde 0.464396 0.294599 10.183 0.6 acid 0.309598 0.196399 12.117 0.7 dihydropyranone 98 0.361197 0.229133 14.883 1 aldehyde 114 0.515996 0.327332 20.95 1.1 C8 acid 0.567595 0.360065 29.817 0.4 unknown 244 0.206398 0.130933 30.633 1.8 acid? 127 0.928793 0.589198 34.55 0.5 unknown aromatic 204 0.257998 0.163666 36.533 6.4 C14 acid 228 3.302374 2.094926 37.9 1.1 unsaturated alcohol? 0.567595 0.360065 38.083 0.8 acid 0.412797 0.261866 39.1 3.5 C15 acid 1.805986 1.145663 39.483 0.6 unsaturated aldehyde? 0.309598 0.196399 39.6 0.5 aldehyde? 0.257998 0.163666 40.567 0.6 aldehyde 237 0.309598 0.196399 40.7 1.5 C16 acid 256 0.773994 0.490998 41.167 3.2 eneoic acid C16? 254 1.651187 1.047463 41.967 21.2 C16 fatty acid 256 10.93911 6.939444 43 0.3 unknown 213 0.154799 0.0982 43.117 1.5 aldehyde 0.773994 0.490998 43.267 3.2 C17 acid 270 1.651187 1.047463 43.483 2.3 C17 acid 270 1.186791 0.752864 44.183 1.3 C17 acid 270 0.670795 0.425532 44.6 0.7 alcohol? alkene? 282 0.361197 0.229133 45.717 1.8 alkyne 284 0.928793 0.589198 45.983 4.9 unsaturated aldehyde or acid 2.52838 1.603928 46.083 6.2 C18 eneoic acid 3.199174 2.02946 46.6 10.7 C18 acid 5.521156 3.502455 49.55 2.8 poly unsaturated alcohol or acid 1.444788 0.91653 49.95 0.8 unknown (cyclic oxygenated) 0.412797 0.261866 50.333 2 unsaturated acid or alcohol 322 1.031992 0.654664 50.5 2.8 eneoic acid 1.444788 0.91653 51.617 1.5 alkane 0.773994 0.490998 52.133 0.8 unknown (unsaturated) 411 0.412797 0.261866 53.067 1.8 alkane 0.928793 0.589198 59.4 2 alkane 1.031992 0.654664 60.2 1.2 unknown 428 0.619195 0.392799 61.183 1.8 alkane 0.928793 0.589198 61.283 0.5 sterol 418 0.257998 0.163666 63.4 0.4 cholestadienol 384 0.206398 0.130933 63.75 4.9 cholestadienol 384 2.52838 1.603928 64.617 54.2 cholesterol 386 27.96698 17.74141 64.683 1.8 cholestanol 388 0.928793 0.589198 65.2 17 methylcholestadienol 398 8.77193 5.564648 66.167 2.7 sterol 398, 426 1.393189 0.883797 66.317 3.6 sterol 400, 403 1.857585 1.178396 Chapter 6: Appendix 6.1 492

Appendix 6.1n Philine angasi continued. Fraction 2 R. t. Area Identity M+/ fragments % fraction 1 % total 66.867 3.5 sterol 412 1.805986 1.145663 68.117 5.5 sterol 414 2.837977 1.800327 68.433 0.8 sterol 412 0.412797 0.261866 70.583 0.4 unknown 400 0.206398 0.130933 71.533 0.9 unknown 430 0.464396 0.294599

Chapter 6: Appendix 6.1 493

Appendix 6.1o Spurilla neopolitana Fraction 1 R. t. Area Identity M+/ fragments % fraction % total 4.517 187767 aldehyde 0.300908 0.297057 4.733 220883 alcohol 0.353978 0.349448 7.383 117148 alkane 0.187736 0.185334 7.467 102099 aldehyde 0.16362 0.161526 9.4 232735 dioxolan 88, 58 0.372971 0.368198 10.017 102847 ester 0.164818 0.162709 10.333 295294 phenol 94 0.473226 0.46717 10.483 125841 dione 0.201668 0.199087 10.7 479981 proply acetate 93, 73 0.769197 0.759354 10.867 1062753 dienal 95, 81 1.703123 1.681328 11.133 84193 aldehyde 0.134924 0.133198 11.417 356083 dienal 0.570644 0.563341 12.167 122020 unstaurated oxygenated 83 0.195544 0.193042 12.283 29480 aromatic 108 0.047243 0.046639 13.083 54570 dienal 0.087452 0.086332 13.2 133524 aldehyde 0.21398 0.211242 13.617 603694 dienone 0.967455 0.955074 13.733 109490 ene 0.175464 0.173219 13.983 189227 furanone 68 0.303247 0.299367 14.067 214518 tri ene 0.343778 0.339378 14.367 122666 unsaturated 0.196579 0.194064 14.517 301987 diene one 0.483952 0.477758 14.633 134472 ene al 0.215499 0.212741 14.783 184429 alkane 0.295558 0.291776 14.967 233625 aldehyde 0.374398 0.369606 15.5 64125 unknown 127, 107 0.102764 0.101449 17 149794 alkene 0.240054 0.236982 17.333 274850 ene ol/nitrite 98, 82 0.440463 0.434826 17.75 144588 unknown 128, 107 0.231711 0.228745 17.95 77704 unsaturated 152, 137 0.124525 0.122932 18.517 371734 alkane 0.595725 0.588102 18.733 121448 aldehyde 0.194627 0.192137 19.117 103524 unknown 139, 127 0.165903 0.16378 19.25 51357 unknown 178, 156 0.082303 0.081249 20.05 245831 C7 dioic acid di me ester 143, 114 0.393958 0.388917 20.75 107811 aldehyde 0.172773 0.170562 20.933 129854 ene ol 87, 67 0.208099 0.205435 21.117 78664 alkane 0.126064 0.12445 21.267 367874 ene al 83, 79 0.589539 0.581995 21.75 950375 ene yne 1.523031 1.50354 21.833 406354 ketone 81, 71 0.651206 0.642872 21.883 176837 yne 0.283392 0.279765 22.183 1593374 unknown 153, 141 2.553474 2.520797 22.567 135409 diene 0.217001 0.214224 22.7 326864 diene 0.523819 0.517115 23.383 159858 unknown 151, 125 0.256182 0.252903 23.867 299083 tetrabutyl urea 156, 106 0.479298 0.473164 24.1 1190277 acetate 135, 121 1.907488 1.883077 24.267 250728 diene one 0.401806 0.396664 Chapter 6: Appendix 6.1 494

Appendix 6.1o Spurilla neopolitana Fraction 1 continued. R. t. Area Identity M+/ fragments % fraction % total 24.74 52124 unknown 183, 148 0.083532 0.082463 24.833 112146 unknown 153, 142 0.17972 0.177421 25.2 72314 unsaturated 150, 135 0.115887 0.114404 25.533 791548 alkane 1.268502 1.252268 26.7 81420 aromatic 173, 158 0.13048 0.12881 28.467 339404 unknown 181, 141 0.543915 0.536954 28.782 391177 alkane 0.626884 0.618861 30.9 3094661 trieneoic acid me ester 4.959374 4.895907 31.217 116337 trieneoic acid me ester 0.186437 0.184051 31.35 1256285 unsaturated alcohol 2.01327 1.987505 32.383 256773 di yne 0.411494 0.406228 32.617 1186340 tri ene ol 1.901179 1.876849 33.217 696183 di ene ol 1.115674 1.101396 34.933 72542 benzoic acid ester 135, 123 0.116253 0.114765 35.1 111928 benzyl phenol 198, 183 0.179371 0.177076 35.2 393929 aldehyde 0.631294 0.623215 35.4 174255 di ene ol 0.279254 0.27568 35.553 233253 alkene 0.373801 0.369018 36.767 56563 unknown 191, 175 0.090645 0.089485 37 143556 alkene 0.230057 0.227113 38.017 759015 aldehyde 96, 82 1.216366 1.200799 38.25 3935618 acid 102, 73 6.307057 6.226343 39.5 332364 di yne 79, 67 0.532633 0.525816 39.6 127979 ene al 0.205094 0.202469 39.7 751492 alcohol 125, 111 1.20431 1.188898 40.267 3795019 diene 95, 81 6.081739 6.003908 40.533 375985 unsaturated 0.602538 0.594827 40.683 914051 aldehyde 1.46482 1.446074 41.083 180313 alcohol 0.288962 0.285264 43.25 2083011 aldehyde 3.338146 3.295427 44.733 835721 ene al 1.339291 1.322152 45.117 196088 alcohol 0.314242 0.310221 45.283 196480 phytol (ene ol) 0.314871 0.310841 45.467 129110 alkane 0.206906 0.204258 45.633 87553 aldehyde 0.140309 0.138513 45.9 679009 aldehyde 1.088151 1.074226 46.4 108533 unknown 0.17393 0.171705 47.3 677519 octadecanoic acid oxy me ester 1.085764 1.071869 47.933 107585 aldehyde 0.172411 0.170205 48.183 141194 aldehyde 0.226272 0.223376 48.5 224439 napthalene deriv. 0.359677 0.355074 48.883 55562 unsaturated alcohol 0.089041 0.087902 49.633 1223593 alkane 1.960879 1.935785 50.317 112828 trieneoic acid me ester 0.180813 0.178499 50.8 5765656 di ene 9.239799 9.121554 51.45 64793 acid me ester 0.103835 0.102506 51.75 2135337 alkane 3.422002 3.378209 51.867 189618 yne 0.303874 0.299985 53.067 125829 triene one 0.201648 0.199068 59.617 357054 trieneol 0.5722 0.564877 Chapter 6: Appendix 6.1 495

Appendix 6.1o Spurilla neopolitana Fraction 1 continued. R. t. Area Identity M+/ fragments % fraction % total 60.7 206504 sterol 368 0.330935 0.3267 61.733 109761 sterol 366 0.175898 0.173647 63.983 2220525 sterol 384 3.55852 3.512981 64.733 5353383 sterol 386 8.579107 8.469318 64.967 1537504 sterol 398 2.46394 2.432408 65.317 274759 sterol 400, 398 0.440317 0.434682 65.883 95648 sterol 398 0.153281 0.15132 66.533 2500120 sterol 314 4.006588 3.955314 66.817 61863 sterol 437, 415 0.099139 0.09787 68.283 76128 sterol 414 0.122 0.120438 68.633 83309 sterol 420, 411 0.133508 0.131799

Fraction 2 R. t. Area Identity M+/ fragments % fraction % total 20.35 16627 benzene acetic acid 91, 136 2.055485 0.026305 30.35 11916 unknown 132, 174 1.473095 0.018852 30.667 11431 acid 1.413138 0.018084 36.45 28624 acid 143, 144 3.538593 0.045285 37.883 12663 eneal 1.565442 0.020033 38.833 28978 ene/eneal 111, 104 3.582356 0.045845 41.117 19926 ene 2.463318 0.031524 41.167 40308 unsaturated ester/aldehdye 162, 125 4.983008 0.063769 41.267 15914 unknown 1.967341 0.025177 41.8 141872 acid 256 17.53868 0.224449 43.1 51175 alcohol/aldehyde 268, 179 6.326422 0.080961 45.9 42509 alcohol/aldehyde 5.255103 0.067251 46.017 45782 propenoic acid ester 5.659722 0.072429 46.483 52097 C18 acid 284 6.440403 0.08242 46.733 24512 unknown (oxy alcohol) 150, 72 3.030254 0.038779 49.567 78444 unknown (yne ol) 106, 91 9.697506 0.124102 50.283 25123 unsaturated (ene al) 112, 98 3.105788 0.039746 50.45 22801 unsaturated 148, 131 2.818735 0.036072 53.667 20341 polyunsaturated 383, 315 2.514622 0.03218 59.55 97554 unknown 247, 217 12.05995 0.154335 64.45 20312 unknown 2.511036 0.032135 Chapter 6: Appendix 6.1 496

Appendix 6.1p Salinator fragilis 1 Fraction 1 R. t. Area Identity M+/ fragments % fraction 1 % total 28.417 48,584 S8 - sulfur molecule 18.4488 8.081144 40.067 24,529 C9? alcohol? 9.314397 4.079993 40.667 20098 C16 fatty acid methyl ester 270 7.631814 3.34297 43.2 108,750 S8 sulfur 41.29564 18.08876 43.567 19,228 alkyne (c12?) 164 7.301449 3.198259 64.317 42,156 unknown (sterol?) 16.0079 7.011953

Fraction 2 R. t. Area Identity M+/ fragments % fraction 1 % total 41.133 103,638 unsaturated fatty acid 30.67511 17.23847 41.65 183,596 C16 fatty acid me ester 256 54.34133 30.53816 45.883 30,823 unknown 9.123091 5.126896 45.9 19,800 unknown 5.860468 3.293402 Chapter 6: Appendix 6.1 497

Appendix 6.1p Salinator fragilis 2 Fraction 1 R. t. Area Identity M+/ fragments % fraction 1 % total 9.333 3.5 dioxolanone 88 0.941873 0.613497 13.333 2.9 ketone 136 0.780409 0.508326 14.717 2.2 alkane 0.592034 0.385627 17.767 0.8 hydropyranone? 114 0.215285 0.140228 18.45 3.6 alkane 0.968784 0.631025 22.033 2.6 alkane 0.699677 0.455741 23.783 5 tetrabutyl urea 156 1.345533 0.876424 25.467 1.4 alkane 0.376749 0.245399 27.317 1.6 unknown 221 0.430571 0.280456 28.4 9.3 unknown 181 2.502691 1.630149 28.883 1.5 sulfur S6 0.40366 0.262927 29.133 3.4 unknown 192 0.914962 0.595968 30.283 0.5 unknown 192 0.134553 0.087642 34.317 1 ene 0.269107 0.175285 34.5 1.3 ene 0.349839 0.22787 34.667 3.4 ene 0.914962 0.595968 34.833 0.8 alkane 0.215285 0.140228 35.083 1.1 hexathiapane 64, 206 0.296017 0.192813 35.383 0.7 acid me ester? 0.188375 0.122699 35.917 0.5 unsaturated 233 0.134553 0.087642 36.933 1.5 unknown 224 0.40366 0.262927 37.917 2.8 aldehyde/alcohol 0.753498 0.490798 39.633 4 aldehyde/alcohol 251 1.076426 0.701139 40.2 15.5 C16 eneoic acid me ester 4.171152 2.716915 40.783 9.7 acid me ester 2.610334 1.700263 41.717 1.9 acid 0.511302 0.333041 43.167 4.2 aldehyde/alcohol 1.130248 0.736196 43.533 59.6 sulfur S8 16.03875 10.44698 43.683 8 unsaturated 2.152853 1.402279 43.8 5.5 unsaturated unknown 202 1.480086 0.964067 45.133 6 C18 eneoic acid me ester 1.614639 1.051709 45.233 11.7 C20 ene ol 296 3.148547 2.050833 45.65 1.7 C18 acid me ester 298 0.457481 0.297984 47.517 2.9 alkene? 0.780409 0.508326 48.583 1.8 unsaturated- tirenoic acid? 0.484392 0.315513 48.817 0.8 unsaturated 281 0.215285 0.140228 50.417 1.9 cyclic ketone 211 0.511302 0.333041 51.383 12.7 hexadione dioctyl ester 259 3.417653 2.226117 53.567 1.6 oxygenated 0.430571 0.280456 59 1.8 unsaturated ketone? 0.484392 0.315513 63.95 27.3 sterol 384 7.346609 4.785276 64.783 80 cholesterol 386 21.52853 14.02279 65.033 1.5 sterol 431 0.40366 0.262927 65.467 26.9 sterol 398 7.238967 4.715162 65.6 2.5 sterol 386 0.672766 0.438212 65.783 1.6 sterol 408 0.430571 0.280456 66.317 2 sterol 314 0.538213 0.35057 66.5 9.3 sterol 400 2.502691 1.630149 66.733 3 sterol 413 0.80732 0.525855 Chapter 6: Appendix 6.1 498

Appendix 6.1p Saliantor fragilis 2 Fraction1 continued. R. t. Area Identity M+/ fragments % fraction 1 % total 67 2.9 sterol 430 0.780409 0.508326 68.3 8.2 sterol 414 2.206674 1.437336 68.583 1 sterol 314 0.269107 0.175285 68.75 0.9 sterol 411 0.242196 0.157756 69.717 0.5 sterol 429 0.134553 0.087642 70.1 1.3 sterol 480 0.349839 0.22787

Fraction 2 R. t. Area Identity M+/ fragments % fraction 1 % total 17.917 0.9 tri/di glycol? 0.452489 0.157756 21.983 0.7 amide 114 0.351936 0.122699 23.317 1.3 unknown 103 0.653595 0.22787 24.667 0.5 unknown 164 0.251383 0.087642 30.35 3.6 unknown 225 1.809955 0.631025 30.638 0.8 C12 acid 200 0.402212 0.140228 30.767 0.9 unknown 127 0.452489 0.157756 36.633 4.3 C14 acid 228 2.16189 0.753725 37.967 1.9 alkane 0.955254 0.333041 38.217 1.2 C15 acid 242 0.603318 0.210342 38.4 1.5 alcohol/aldehyde 0.754148 0.262927 39.25 3 C15 acid 242 1.508296 0.525855 42.183 45.8 C16 eneoic acid 254 23.02665 8.028046 42.617 38.9 C16 acid 256 19.55757 6.81858 44 4.9 C17 eneoic acid 268 2.46355 0.858896 46.65 22.1 C18 eneoic acid 282 11.11111 3.873795 46.917 3.3 C18 acid 284 1.659125 0.57844 50.2 24.5 unsaturated unknown 12.31775 4.294479 50.817 2.7 unsaturated 1.357466 0.473269 53.583 12.8 eneal/eneol? 6.435395 2.243646 54.017 3.2 acetic acid ester? 1.608849 0.560911 57.467 1.3 ene al? 0.653595 0.22787 57.55 1.4 ene ol? 0.703871 0.245399 60.467 17.4 unsaturated unknown 8.748115 3.049956 Chapter 6: Appendix 6.1 499

Appendix 6.1q Salinator solida Fraction 1 R. t. Area Identity M+/ fragments % fraction 1 % total 5.55 67 C6 ketone 59, 101 6.405966 3.77833 23.783 3.2 urea tetra butyl 0.305957 0.299261 26.633 1.3 tetramethyl dihydro quinoline 173 0.124295 0.121575 26.95 10.3 dimethylphthalate 163 0.984798 0.963247 27.317 1 unknown 180 0.095611 0.093519 27.9 1.3 alcohol? 177 0.124295 0.121575 28.4 9.6 unknown 181 0.91787 0.897784 28.717 1.9 alkane 0.181662 0.177686 29.133 1.1 unknown 180 0.105173 0.102871 29.383 2.1 Benzofuranone tetrahydro trimethyl 111 0.200784 0.19639 29.433 2.9 C12 acid me ester 214 0.277273 0.271205 31.333 6.9 di ester? 0.659719 0.645282 34.35 9.9 C14 acid me ester 242 0.946553 0.925839 34.55 9.2 alkane 0.879625 0.860376 34.75 14.6 alkane 1.395927 1.365379 35 2.3 ene oic acid 0.219906 0.215094 35.133 1.2 unknown 149 0.114734 0.112223 35.167 1.4 alkene? 0.133856 0.130927 35.45 48.8 C14 acid me ester 242 4.665838 4.563733 35.967 1.6 alkan? 0.152978 0.149631 36.533 1.1 aldehyde? 0.105173 0.102871 36.833 9 ene one? 0.860503 0.841672 36.933 1.8 aldehyde/alcohol 0.172101 0.168334 37.183 16.6 C15 fatty acid me ester 256 1.58715 1.552417 37.383 12 fatty acid me ester 1.147337 1.122229 37.95 2 aldehyde 0.191223 0.187038 38.15 6.4 fatty acid me ester 0.611913 0.598522 38.583 3.9 ketone 0.372885 0.364725 39.817 9.9 trieneoic acid 0.946553 0.925839 40.1 6.3 C16? eneoic acid me ester 0.602352 0.58917 40.367 121.2 C16 eneoic acid me ester 268 11.58811 11.33452 40.533 22.8 C16 eneoic acid me ester 2.179941 2.132236 40.65 2.9 alcohol/aldehyde 0.277273 0.271205 40.983 146 C16 acid me ester 270 13.95927 13.65379 41.767 4 C18 eneoic acid 282 0.382446 0.374076 41.9 5.9 C17 acid me ester 284 0.564107 0.551763 41.967 3.7 eneoic acid 0.353762 0.346021 42.083 9.9 alcohol/alkene 0.946553 0.925839 42.267 2.1 alcohol/aldehyde 0.200784 0.19639 42.4 12.9 C17 acid me ester 284 1.233388 1.206397 42.517 3.8 ethyl ester 0.363323 0.355373 42.6 5.9 C17 fatty acid me ester 284 0.564107 0.551763 42.683 6.6 eneoic acid me ester 0.631035 0.617226 42.867 5.7 eneoic acid me ester 0.544985 0.533059 43.167 3.5 aldehyde/alcohol 0.33464 0.327317 43.3 6.7 C17 acid me ester 284 0.640597 0.626578 43.683 1.8 unsaturated unknown 271 0.172101 0.168334 43.8 0.8 unsaturated alcohol? 202 0.076489 0.074815 44.4 2.3 yne/yneol? 0.219906 0.215094 Chapter 6: Appendix 6.1 500

Appendix 6.1q Salinator solida Fraction 1 continued. R. t. Area Identity M+/ fragments % fraction 1 % total 44.533 6.5 unsaturated 0.621474 0.607874 44.85 24.7 unsaturated alcohol/aldehyde 294 2.361602 2.309922 44.917 28.3 C18 dieneoic acid me ester 294 2.705804 2.646591 45.15 62.6 C18 eneoic acid me ester 296 5.985276 5.854297 45.333 99.5 C18 eneoic acid me ester 9.513338 9.305153 45.45 3.1 C18 eneoic acid me ester 296 0.296395 0.289909 45.717 19.3 alkene 298 1.845301 1.804919 45.8 8 C18 acid me ester 0.764891 0.748153 46.567 2.9 eneoic acid ester 0.277273 0.271205 47.633 22 C19 eneoic acid me ester 310 2.103452 2.057421 48.5 6.9 polyunsaturated acid me ester 0.659719 0.645282 48.65 14.9 polyunsaturated ester 1.42461 1.393435 49.417 4.8 C20 dieneoic acid me ester 322 0.458935 0.448892 49.683 3.7 C20 eneoic acid me ester 324 0.353762 0.346021 52.517 2.3 polyunsaturate 0.219906 0.215094 53.567 16.3 oxygenated 1.558466 1.524362 57.55 13.4 alcohol 1.281193 1.253156 58.767 23.1 acid hydroxy me ester 383 2.208624 2.160292 61.283 23.3 alcohol 2.227746 2.178996 61.783 0.2 sterol 410 0.019122 0.018704 63.117 2.8 unsaturated 0.267712 0.261854 63.85 5.8 sterol 384 0.554546 0.542411 64.633 37 sterol 386 3.537623 3.460208 65.217 4.7 sterol 398 0.449374 0.43954 67.05 5.3 sterol 413 0.506741 0.495651 67.933 5 sterol 424 0.478057 0.467596 68.4 20.5 sterol 414 1.960034 1.917142 68.633 1.7 sterol 416 0.162539 0.158983 68.75 5.3 sterol 426 0.506741 0.495651 70.267 3.9 sterol 426 0.372885 0.364725 fraction 2 R. t. Area Identity M+/ fragments % fraction 1 % total 4.167 2.1 formamide dimethyl 8.974359 0.19639 5.533 45.6 hydroxy methyl pentanone 194.8718 4.264472 30.65 0.7 acid 2.991453 0.065463 30.683 1.1 unknown 127 4.700855 0.102871 30.733 0.7 unknown 127 2.991453 0.065463 41.267 4.7 eneoic acid me ester 20.08547 0.43954 41.817 5.5 C16 fatty acid 256 23.50427 0.514355 43.4 1.2 unknown 230 5.128205 0.112223 45.7 0.9 eneol/ triene 280 3.846154 0.084167 45.917 2.6 eneol/ eneoic acid 11.11111 0.24315 46.033 2.6 unsaturated acid 11.11111 0.24315 46.483 1.3 acetic acid ester 5.555556 0.121575 49.5 0.7 dienol? 2.991453 0.065463 57.433 0.4 unknown 68, 97 1.709402 0.037408 57.533 0.4 alkane 226 1.709402 0.037408 61.15 0.6 unknown 421, 436 2.564103 0.056111 Chapter 6: Appendix 6.1 501

Appendix 6.1r Siphonaria denticulata 1 Fraction 1 R. t. Area Identity M+/ fragments % fraction 1 % total 4.483 49.7 hexanal 1.464263 0.727875 9.317 29.3 C7enal 0.863237 0.429109 10.767 55.7 heptadienal 110 1.641035 0.815747 13.1 24.1 C7ene 98 0.710035 0.352953 18.45 34.1 C12 ene 170 1.004655 0.499407 20.65 24.3 C10 enal 0.715927 0.355882 21.633 36 C10 unsaturated (ene yne) 136 1.060633 0.527233 21.8 109.7 decadienal 152 3.231984 1.606596 22.05 39.1 unknown (subst. alkane) 1.151965 0.572634 22.633 170.8 decadienal 152 5.032114 2.501428 25.45 54.8 alkane 1.614519 0.802566 25.783 22.4 unknown 127 0.659949 0.328056 28.383 90.9 unknown 2.678098 1.331263 28.467 29 alkene (C10?) 140 0.854399 0.424716 28.717 206.1 C12 alkane 170 6.072123 3.018409 31.133 31.6 C11 unsaturated alcohol 0.931 0.462793 31.217 25.8 alkyne (eneyne?) 150 0.76012 0.37785 32.15 35.7 alkyne or alcohol 166 1.051794 0.522839 35.117 88.1 unsaturated alcohol? 2.595604 1.290256 36.883 45.6 C16 alcohol 1.343468 0.667829 37.1 60.5 alcohol/aldehyde 1.782452 0.886044 37.917 124.1 aldehyde 3.656237 1.817489 39.517 38.1 ene al? 1.122503 0.557988 40.6 143.3 aldehyde 4.221908 2.09868 43.133 134.3 aldehyde/alcohol 3.95675 1.966872 48.5 342 unknown 10.07601 5.008714 59.533 134.2 unsaturated alcohol? 3.953804 1.965408 64.9 1159.1 sterol 386 34.14943 16.97544 68.1 30.4 unknown 314 0.895646 0.445219 68.483 25.4 unknown 314 0.748335 0.371992

Fraction 2 R. t. Area Identity M+/ fragments % fraction 1 % total 10.4 21.3 dienal 0.620286 0.311946 10.5 15.1 pentanoic acid 0.439733 0.221145 10.75 11 heptadienal 110 0.320335 0.161099 13.1 18.3 C7 diene ether 0.532922 0.26801 13.283 40.3 ketone 1.173593 0.590208 14.483 17.9 C7 ene al 0.521273 0.262152 17.617 93.1 unsaturated 166 2.711203 1.363483 20.1 18.6 C12 ene 168 0.541658 0.272404 21.783 31.6 alkyne 124 0.920236 0.462793 22.6 40.6 C10 diene al 152 1.182329 0.594602 23.867 49.4 alkane 1.438598 0.723481 24.2 15.8 C8 ene one 126 0.460118 0.231397 24.65 20.6 unsaturated dione or dioic acid 127 0.599901 0.301694 25.85 100.4 ketone 142 2.923789 1.470394 27.183 19.1 ketone 0.556219 0.279726 30.733 37.1 unknown 127 1.080404 0.543343 Chapter 6: Appendix 6.1 502

Appendix 6.1r Siphonaria denticulata Fraction 2 continued. R. t. Area Identity M+/ fragments % fraction 1 % total 35.017 14.8 unknown 166 0.430997 0.216751 35.083 16 C17 alcohol 242 0.465943 0.234326 36.55 66.2 C14 acid 228 1.927837 0.969523 36.617 74.3 C14 acid 228 2.163721 1.08815 37.9 43.7 C14 aldehyde? 212 1.272605 0.640002 39.167 45.9 C15 acid 242 1.336673 0.672222 39.5 17.7 ene al? 0.515449 0.259223 39.8 25.7 ene al 0.74842 0.376386 40.567 23.1 aldehyde 0.672705 0.338308 41.233 46.4 eneoic acid 1.351233 0.679545 41.367 106.9 C18 ene al? 266 3.113078 1.565589 41.967 144.8 C16 acid 256 4.21678 2.120648 42.25 350.7 C16 acid 256 10.21288 5.136129 43.133 67.2 Aldehyde 1.956959 0.984168 43.633 49.6 fatty acid 1.444422 0.72641 44.25 57.1 C17 acid 270 1.662832 0.83625 46.05 102.9 unsaturated alcohol 2.996593 1.507008 46.183 148.5 ene al 4.324529 2.174836 46.267 186.8 C18 eneoic acid 282 5.439879 2.735754 46.35 250.9 ene al? 7.306561 3.674521 46.8 121.7 C18 acid 284 3.544075 1.782341 49.817 59.8 polyunsaturated 1.74146 0.875793 49.717 53 unsaturated 1.543435 0.776204 49.95 45.7 unknown 166 1.330848 0.669293 50.267 32.2 unknown 103 0.937709 0.471581 50.467 73.4 ene ol 308 2.137511 1.07497 50.6 84.8 ene al 292 2.469495 1.241927 50.717 37.8 unknown 103 1.100789 0.553595 51.383 16.9 oxygenated 103 0.492152 0.247507 51.633 351.9 alkane C23 324 10.24782 5.153703 53.95 142.6 C16 acid dihydroxy propyl ester 4.152713 2.088429 60.2 24.7 sterol cholestadienol 0.719299 0.36174

Chapter 6: Appendix 6.1 503

Appendix 6.1r Siphonaria denticulata 2 Fraction 1 R. t. Area Identity M+/ fragments % fraction % total 27.333 49126 unknown 180, 168 0.585384 0.175191 28.4 99383 unknown 141 1.184244 0.354415 28.717 289785 alkane 3.453067 1.033419 31.373 146842 unknown 149 1.749764 0.523662 35.117 183040 aldehyde 198, 183 2.181098 0.652749 37.933 58380 unknown 0.695654 0.208192 38.033 61235 alcohol/ alkene 143, 111 0.729674 0.218374 38.567 84946 ketone/ aldehyde 126, 109 1.012213 0.302931 39.683 108813 ene 1.296612 0.388044 39.917 75090 ene al 0.89477 0.267783 40.583 52365 aldehyde/ alcohol 0.623979 0.186742 40.75 eneoic acid me ester 2.0112 0.601903 41.75 242021 acid 2.883913 0.863085 43.133 98785 alcohol 1.177118 0.352283 43.317 116210 alcohol 1.384754 0.414423 43.533 156049 diyne/ ene yne 1.859474 0.556495 43.65 52343 unknown 167, 108 0.623717 0.186663 43.767 74025 unsaturated 303 0.882079 0.263985 44.717 254678 ene 3.034733 0.908222 44.833 112819 ene one 202, 187 1.344347 0.40233 45.033 82473 phenyl ester 117, 217, 232 0.982745 0.294112 45.233 235797 ene ol 123, 71 2.809748 0.840889 46.35 35885 unknown 209, 179 0.427604 0.127972 46.5 94691 acid 1.128334 0.337683 47.5 931190 unsaturated 11.09602 3.32077 48.517 1508002 unknown 216, 43 17.96929 5.377772 48.85 55515 unknown 210 0.661515 0.197975 49.8 40981 unknown 178, 161 0.488328 0.146145 51.35 85983 hexanedioic acid di octyl ester 147, 129 1.02457 0.306629 53.583 130589 alcohol 1.556093 0.465701 59.533 615063 trienol 7.329067 2.193411 60.65 42949 yne 0.511779 0.153163 61.767 73532 unknown 177, 161 0.876204 0.262227 64.317 84740 unsaturated oxygenated 1.009759 0.302196 64.583 1469018 cholesterol 386 17.50476 5.238749 64.683 91981 sterol 1.096042 0.328019 65.233 78881 sterol 0.939943 0.281302 68.2 250118 sterol 314 2.980396 0.89196 Chapter 6: Appendix 6.1 504

Fraction 2 R. t. Area Identity M+/ fragments % fraction % total 14.917 67249 heptenal 0.342247 0.23982 17.617 338959 octadiene diethyl 166, 108 1.725045 1.208781 18.667 39896 ene 142, 100 0.20304 0.142275 21.2 61345 acid 0.3122 0.218766 22 85933 amine 114, 72 0.437334 0.306451 23.5 145319 unknown 69 0.739564 0.51823 24.25 80463 unknown 126, 97 0.409496 0.286944 24.517 29430 acid 0.149776 0.104952 24.883 50613 unknown 127 0.257582 0.180494 25.9 313037 ketone/ aldehyde 86, 57 1.593122 1.116339 27.55 265597 benzoic acid hydroxy me ester 152, 121 1.351688 0.947161 27.7 55698 isoindoledione hydroxy methyl 147, 104 0.283461 0.198628 Appendix 6.1r Siphonaria denticulata 2 Fraction 2 continued. R. t. Area Identity M+/ fragments % fraction % total 27.9 96283 unknown 163, 91 0.490008 0.34336 29.133 63763 unknown 99 0.324505 0.227389 30.167 94627 unknown 136, 93 0.48158 0.337455 30.883 241335 acid 1.228213 0.860639 35.1 98246 benzaldehyde 166 0.499998 0.350361 37.75 659123 acid 3.354438 2.350536 37.067 165566 diol/ ester 180, 69 0.842606 0.590434 37.933 80522 alkane 0.409796 0.287154 38.25 125299 ether 0.637677 0.446836 38.417 186097 eneol 0.947093 0.663651 39.25 251588 acid 1.280393 0.897202 39.567 48379 unsaturated oxygenated 0.246213 0.172527 41.033 87437 unknown 172, 116 0.444988 0.311814 41.317 338393 ene al 1.722164 1.206763 42.333 2775214 acid 256 14.12374 9.896849 43.15 77955 aldehyde/ alcohol 0.396732 0.278 43.417 119651 C17 acid 270 0.608933 0.426694 43.633 214354 eneoic acid 167 1.0909 0.76442 43.75 73401 unsaturated acid 285, 180 0.373556 0.261759 44.283 284577 acid 270 1.448282 1.014846 46.283 1496120 eneoic acid 282 7.614119 5.335399 46.817 1303358 acid 284 6.633106 4.647979 47.933 120196 ene one 200, 185 0.611707 0.428638 48.517 416422 unknown 234, 216 2.119273 1.485026 49.567 254272 trienoic acid 1.294052 0.906774 49.65 271816 unsaturated (ene yne) 1.383338 0.969339 49.833 111321 unsaturated (trieneol) 0.56654 0.396988 50 164364 unsaturated (trieneol) 0.836488 0.586148 50.1 96572 unsaturated 0.491478 0.344391 50.3 125373 unsaturated (ene ol) 0.638054 0.4471 50.467 230874 eneoic acid 1.174974 0.823333 50.583 124200 unsaturated 0.632084 0.442917 50.967 248099 acid 1.262636 0.88476 52.3 132962 dehydroabietic acid 300, 285 0.676676 0.474163 53.15 63250 unknown 302, 259 0.321895 0.225559 54.117 1776673 unknown 9.041922 6.335895 Chapter 6: Appendix 6.1 505

Appendix 6.1r Siphonaria denticulata 2 Fraction 2 continued. R. t. Area Identity M+/ fragments % fraction % total 55.033 116186 ether 0.591299 0.414338 59.7 3806581 unknown 173, 217 19.37262 13.57487 64.55 944731 cholesterol 386 4.807966 3.369059 64.7 61618 sterol 384 0.313589 0.219739 68.183 168948 sterol 314 0.859818 0.602495 Chapter 6: Appendix 6.1 506

Appendix 6.1s Siphonaria zelandica Fraction 1 R. t. Area Identity M+/ fragments % fraction 1 % total 22.1 54192 141 0.380549 0.191158 24.683 30181 aromatic pyranone 183 0.211938 0.106461 25.817 143563 127 1.008132 0.506407 26.667 188099 quinoline 158 1.320874 0.663504 26.933 20022 quinoline 163 0.140599 0.070626 27.25 86323 quinoline 180 0.60618 0.304498 27.983 58846 alkene 111 0.41323 0.207575 28.433 981610 141 6.89309 3.462551 28.733 270432 alkane 1.899035 0.953927 28.983 17746 unknown 206 0.124616 0.062598 30.3 29024 unknown 192, 220 0.203813 0.10238 31.35 292331 unknown 71, 149 2.052815 1.031174 34.317 88971 alkene 199 0.624775 0.313838 34.517 38611 ene/alcohol 152 0.271135 0.136197 34.85 46391 unknown 156 0.325768 0.163641 35.133 94402 unknown 198 0.662912 0.332996 35.4 64056 me ester 101,111 0.449816 0.225952 36.767 48791 unknown 191 0.342622 0.172106 37.667 79421 alkane 247 0.557712 0.280151 37.95 66213 aldehyde 110 0.464963 0.233561 40.783 294568 me ester 143 2.068524 1.039065 41.8 239583 acid 256 1.682407 0.84511 43.167 168916 aldehyde 1.186167 0.595838 44.733 1570367 hexadecanol 242 11.02748 5.539345 45.067 249859 phenylester 117, 232 1.754567 0.881358 45.15 116119 alkene 0.815414 0.409601 47.233 259930 unknown 234 1.825288 0.916882 48.567 3385649 unsat. Ketone 23.7748 11.94261 48.867 152702 unknown 210 1.072308 0.538644 49.333 79.367 unsat. 155 0.000557 0.00028 49.467 393171 unknown 274 2.760937 1.386879 51.367 172913 dioic acid dioctyl ester 241, 259 1.214235 0.609937 53.6 346882 alcohol 2.435885 1.223599 59.583 1371174 diene 9.628697 4.836707 64.383 269501 unknown diene? 1.892498 0.950643 64.717 2385261 cholesterol 16.74985 8.413818 65.267 46191 sterol 351 0.324364 0.162935 68.217 62856 sterol 380 0.441389 0.22172 68.617 55548 sterol 314 0.390071 0.195941 Chapter 6: Appendix 6.1 507

Fraction 2 R. t. Area Identity M+/ fragments % fraction 1 % total 14.917 63799 alcohol 0.452192 0.225046 15.8 46100 me ester 196 0.326746 0.162614 17.633 226254 unknown 166 1.603634 0.798093 21.217 64220 acid 0.455176 0.226531 22 247368 dibutylformamide 157 1.753285 0.872571 23.8 76.76 tetrabutyl urea 0.000544 0.000271 24.483 33785 acid 0.23946 0.119174 24.9 44214 unknown 127 0.313378 0.155961 27.567 114816 benzoic acid hydroxy me ester 152 0.813788 0.405004 27.717 92593 isoindole dione 147 0.656277 0.326614 28.217 21713 unknown 113 0.153896 0.076591 Appendix 6.1s Siphonaria zelandica Fraction 2 continued. R. t. Area Identity M+/ fragments % fraction 1 % total 29.133 50196 unknown 155 0.355777 0.177062 30.45 1152716 unknown 174 8.170172 4.066114 30.967 318174 acid 2.25514 1.122333 33.167 59321 unknown 125 0.420453 0.20925 35.117 142477 unknnow 165,166 1.009842 0.502576 36.733 604597 acid 4.285237 2.132668 38.017 126859 acid 0.899146 0.447485 38.45 98635 aldehyde 0.699101 0.347927 39.25 212324 acid 1.504901 0.748956 41.05 159464 unknown 172 1.130242 0.562497 42.333 2568426 acid 18.20438 9.059918 43.167 79907 aldehyde 0.566361 0.281866 43.45 278497 acid 1.973919 0.982376 43.667 306063 acid 2.1693 1.079613 44.317 406774 acid 2.883116 1.434863 46.383 1893780 unsat. acid 13.42265 6.680158 46.917 1594625 acid 284 11.30232 5.624913 47.117 735624 amide 5.213925 2.594855 47.967 237312 acetate 200 1.68201 0.837099 48.55 1205976 acetate 234 8.547666 4.253984 49.633 343833 unsat alcohol 2.437005 1.212844 49.75 418620 unsat 2.967077 1.476649 49.9 159695 unsat 1.13188 0.563311

Chapter 6: Appendix 6.1 508

Appendix 6.1t Isidorella hainesi Fraction 1 R. t. Area Identity M+/ fragments % fraction % total 12.083 2 benzyl alcohol M+ 108 0.099542 0.068171 14.467 4 C7 unsat. alcohol M+ 100 0.199083 0.136342 22.55 2.3 C7 diene M+ 96 0.114473 0.078397 22.567 2.1 C10 enal M+ 154 0.104519 0.07158 24.117 1.8 C9 yne M+ 124 0.089587 0.061354 25.383 3.4 unknown (unsat. amine) 95, 124, 155 0.169221 0.115891 25.733 25.5 unknown 167 1.269156 0.86918 27.217 32.8 unsat. ketone alcohol 180, 221 1.632482 1.118004 27.817 2.8 unsat. alcohol 166 0.139358 0.095439 28.333 176.4 unknown (cyanide) 141, 181 8.77957 6.01268 29.283 3.8 unknown (benzoic acid ethoxy ethyl ester) 166, 194 0.189129 0.129525 30.483 3.7 unknown (pyridyl ketone) 162, 204 0.184152 0.126116 30.967 2.8 ketone 142 0.139358 0.095439 31.283 10.4 unknown (aromatic ketone) 159, 173 0.517616 0.354489 31.75 3.8 ketone 142 0.189129 0.129525 32.1 5.2 unknown 193, 195 0.258808 0.177245 33.967 6.3 alcohol 200 0.313556 0.214739 34.2 3.1 aldehyde 156 0.154289 0.105665 34.483 5.2 unsat. phenyl ketone) 147, 161 0.258808 0.177245 35.05 24.1 enal 170 1.199476 0.82146 35.333 8.8 C15 me ester M+ 242 0.437983 0.299952 35.817 1.5 alkane 198 0.074656 0.051128 36.267 4.1 unknown 161, 203, 205 0.20406 0.13975 36.833 10.4 dienal 126 0.517616 0.354489 37,050 12.8 unknown 0.637066 0.436294 37.25 2.4 unknown 226 0.11945 0.081805 38.1 174.6 ethoxy ketone 228 8.689983 5.951326 38.5 8.2 ketone 198 0.408121 0.279501 39.422 12.3 enal 135 0.612181 0.419251 39.517 17.8 ketone 236 0.88592 0.606722 39.75 4.1 diene 154 0.20406 0.13975 40.1 6.8 unknown 0.338441 0.231781 40.317 55.5 ene 168 2.76228 1.891744 40.517 9 unsat. ketone 208 0.447937 0.306769 40.7 87.1 C16 me ester M+ 270 4.335037 2.968846 41.95 12.6 unsat.oxy 156 0.627112 0.429477 42.15 29.6 unsat. oxy 136, 163 1.473216 1.00893 44.567 17.3 alcohol 166 0.861035 0.589679 44.917 7.4 C18 eneoic acid me ester 279 0.368304 0.252233 45.05 10.7 eneoic acid me ester 0.532548 0.364715 45.3 14 ene 156 0.696791 0.477197 45.567 35.7 C18 me ester 298 1.776818 1.216852 47.283 10.2 unknown 193, 316 0.507662 0.347672 51.167 11.1 unknown 283, 285, 313 0.552456 0.378349 51.283 72.6 hexanedioic acid dioctyl ester 3.61336 2.474606 51.5 12.9 unknown (oxy phenyl) 191, 279 0.642043 0.439703 53.417 13.4 unsat. alcohol 284 0.666929 0.456746 61.117 17 alcohol 0.846104 0.579453 63.667 13.8 unknown 0.686837 0.47038 Chapter 6: Appendix 6.1 509

Appendix 6.1t Isidorella hainesi Fraction 1 continued. R. t. Area Identity M+/ fragments % fraction % total 64.583 699 cholesterol 301, 386 34.78979 23.82575 65.1 30.7 substituted cholestadienol 300, 398 1.527964 1.046424 65.35 6.4 sterol 368, 386 0.318533 0.218147 66.267 72 sterol 3.583498 2.454155 66.85 95.4 sterol M+ 414 4.748135 3.251755 68.083 90.5 sterol? 4.504258 3.084737

Fraction 2 R. t. Area Identity M+/ fragments % fraction % total 4.45 8 C6 aldehyde 29.62963 0.272684 13.083 4.9 unknown (amine?) 18.14815 0.167019 14.833 2.9 C8 enal 112 10.74074 0.098848 20.633 2.9 alkene or aldehyde 10.74074 0.098848 30.583 2.4 unknown 8.888889 0.081805 41.583 3.3 C16 fatty acid me ester 256 12.22222 0.112482 51.517 2.6 unknown 9.62963

Chapter 6: Appendix 6.1 510

Appendix 6.1u Bembicium nanum 1 Fraction 1 R. t. Area Identity M+/ Fragments % fraction 1 % total 9.25 106,774 C5 enal 84 0.387867 0.360751 10.717 69,075 C7 dienal 110 0.250922 0.23338 11.267 35,889 C7 dienal 110 0.13037 0.121256 18.417 55,784 alkane 0.202641 0.188474 20.6 122,342 C8 alkene 112 0.444419 0.41335 21.55 39,439 unsat. alkane or alcohol 0.143266 0.13325 21.733 84,916 C10 dienal 152 0.308466 0.286901 21.983 96,925 unknown 0.352089 0.327475 22.533 99,012 C10 dienal 152 0.359671 0.334526 25.733 637,502 unknown 2.315788 2.153893 26.983 44,624 unsat. or cyclo C7 aldehyde 112 0.162101 0.150769 27.217 49,303 unknown 167 0.179098 0.166577 27.567 20,326 unknown 218 0.073836 0.068674 27.817 39,420 C9 unsat. alcohol 142 0.143197 0.133186 28.3 83,008 unknown 0.301535 0.280455 28.417 50,524 C10 alkene 240 0.183533 0.170703 28.683 135,496 C11 alkene 156 0.492202 0.457793 29.4 138,769 C7 fatty acid methyl ester 144 0.504092 0.468851 31.267 58,289 alkyl lactone 331 0.21174 0.196938 35.05 74,927 aldehyde (C13enal?) 0.27218 0.253152 35.35 3,261,834 C14 fatty acid methyl ester 242 11.84893 11.02058 37.867 411,112 C13 eneol 198 1.493404 1.389001 38.083 322,373 C17 ester 270 1.171051 1.089184 39.983 60,001 unsaturated fatty acid 0.217959 0.202722 40.117 344,649 C16 eneoic acid methyl ester 268 1.251971 1.164446 40.367 78,723 C16 enoic acid me ester 268 0.285969 0.265977 40.533 180,522 C16 ene ol 0.655764 0.60992 40.767 6,229,216 C16 fatty acid me ester 270 22.62823 21.04631 42.283 125,759 C17 fatty acid methyl ester 284 0.456832 0.424895 42.467 98,727 C17 acid me ester 284 0.358635 0.333563 43.083 1,114,672 C18 aldehyde 268 4.049154 3.76608 43.183 176,985 C17 acid me ester 284 0.642915 0.597969 44.443 65,051 C18 trienoic methyl ester 292 0.236304 0.219784 44.783 1,576,370 C18 dienoic acid me ester 294 5.726316 5.325994 44.983 2,454,574 C18 enoic acid me ester 296 8.916477 8.293133 45.083 337,359 C18 enoic acid me ester 296 1.225489 1.139816 45.583 1,072,628 C18 acid me ester 298 3.896425 3.624029 47.317 267,014 C20 enol 296 0.969954 0.902145 48.4 171,950 C20 trieneoic acid me ester 320 0.624625 0.580958 48.517 555,167 C20 trienoic acid me ester 320 2.016698 1.875712 48.733 138,727 unknown 0.503939 0.468709 48.883 571,581 C20 dienoic acid me ester 322 2.113478 1.921654 49.317 782,466 C20 dienoic acid me ester 322 2.842383 2.643674 49.5 1,843,849 long chained me ester 6.69796 6.22971 49.6 450,158 (C22?) unsaturated me ester 1.635242 1.520924 51.283 46,067 C(6)? dioic dioctyl ester 0.167343 0.155644 52.617 97,949 trienoic acid me ester 0.355809 0.330935 52.75 393,396 unsaturated me ester 318 1.429049 1.329145 52.983 83,042 C18 dieneol? 266 0.301658 0.280569 Chapter 6: Appendix 6.1 511

Appendix 6.1u Bembicium nanum 1 Fraction 1 contd. R. t. Area Identity M+/ Fragments % fraction 1 % total 53.117 1,299,876 unsaturated ester 350 4.721925 4.391819 64.367 727,345 cholesterol 386 2.642151 2.45744 66.033 28,215 sterol 0.102494 0.095328 67.683 114,307 460 0.415231 0.386203

Appendix 6.1u Bembicium nanum 1 Fraction 2 R. t. Area Identity M+/ Fragments % fraction 1 % total 36.383 156,502 long alkyl glycine (eg. sarcosine, N lauroyl) 7.563585 0.528765 41.75 933,644 C16 fatty acid me ester 45.12208 3.154451 45.65 87,350 unsaturated fatty acid 4.221538 0.295125 45.833 194,349 fatty acid me ester 9.392693 0.656636 46.417 129,941 fatty acid me ester 6.279919 0.439024 49.433 54,549 fatty acid or unsaturated aldehyde 2.636299 0.184302 49.833 80,288 unsaturated aldehyde 3.880239 0.271265 50.267 171,915 unsaturated aldehyde 8.30848 0.58084 64.367 260,613 cholesterol 12.59517 0.880519

Chapter 6: Appendix 6.1 512

Appendix 6.1u Bembicium nanum 2 fraction 1 R. t. intensity identity M+/ fragments % fraction % total 22.067 59263 unknown 141 1.835957 0.273871 25.8 40933 unknown 127 1.268097 0.189163 28.4 316896 unknown 141 9.817383 1.464463 28.717 128969 alkane 3.995437 0.596001 32.467 39631 trophenyl trioxolane 182, 105 1.227762 0.183146 35.117 34058 aldehyde 1.055111 0.157391 36.467 61047 alkane 1.891225 0.282115 37.917 42474 ether? 83, 97 1.315837 0.196284 38.133 482598 acid 228 14.9508 2.230218 40.6 51440 aldehyde 1.593602 0.237718 40.75 63920 C16 me ester 270, 228 1.980231 0.295392 41.2 74238 eneoic acid 2.29988 0.343074 41.75 259980 acid 256 8.054135 1.201439 42.583 49898 C18 ene al 266 1.545831 0.230592 43.133 60275 aldehyde 1.867309 0.278547 44.7 211674 alkene/ alcohol 224 6.557624 0.978204 45.05 40437 aldehyde 1.252731 0.18687 45.833 28156 unsaturated oxygenated 0.872268 0.130117 46 38180 acid 1.18281 0.17644 46.467 46040 acid 242 1.426311 0.212763 59.567 70607 sterol 2.187393 0.326294 60.65 54662 sterol 368 1.693419 0.252608 61.05 25594 sterol 0.792898 0.118277 61.667 68553 sterol 366, 350 2.12376 0.316802 62.3 52882 sterol 366 1.638275 0.244382 62.917 54889 alkane 1.700452 0.253657 64.55 770613 cholesterol 286 23.87346 3.561214

Fraction 2 R. t. intensity identity M+/ fragments % fraction % total 21.367 91322 acid 0.496015 0.422024 22.017 133168 amine 114, 72 0.723301 0.615406 24.533 55576 acid 0.301861 0.256832 24.883 139469 unknown 0.757525 0.644525 27.517 58878 benzoic acid hydroxy me ester 151, 121 0.319795 0.272091 30.933 357148 unknown 127, 99 1.939847 1.650479 33.683 57101 acid 282 0.310144 0.263879 34.5 165141 alcohol 0.896962 0.763162 36.917 831549 acid 228 4.516552 3.842816 37.183 223875 unknown 103, 69 1.215975 1.034588 38.067 173150 ether 0.940463 0.800174 38.317 153318 acid 0.832746 0.708525 38.5 110834 ester 0.601994 0.512194 39.317 346614 acid 1.882631 1.601798 41.05 104803 unknown 273, 172 0.569237 0.484323 41.6 1142624 eneoic acid 6.206154 5.280379 42.383 2596975 acid 256 14.10545 12.00133 43.117 67972 ene/ alcohol 257 0.369189 0.314117 43.617 525943 acid 270 2.856656 2.430527 Chapter 6: Appendix 6.1 513

Appendix 6.1u Bembicium nanum 2 Fraction 2 continued. R. t. intensity identity M+/ fragments % fraction % total 43.767 253923 acid 1.379181 1.173448 43.9 152666 ene 0.829204 0.705511 44.133 155122 eneoic acid 268 0.842544 0.716861 44.483 848616 acid 270 4.609252 3.921688 46.433 1239545 eneoic acid 6.73258 5.728278 47.033 2493520 acid 284 13.54354 11.52324 48.417 156636 ene 0.850767 0.723858 48.85 69621 yne ol/ ene al 210, 131 0.378146 0.321738 49.75 530100 yne ol/ ene yne 2.879234 2.449738 50.133 490926 ene ol 2.666461 2.268704 50.35 233281 ene ol 1.267064 1.078056 50.633 727088 eneoic acid 292 3.949173 3.360073 50.75 830996 eneoic acid 292 4.513549 3.840261 51.083 327238 acid 1.777391 1.512257 64.683 2267208 cholesterol 386 12.31432 10.47739 66.25 67201 sterol 384, 314 0.365002 0.310554 68.15 102938 sterol 382, 314 0.559107 0.475705 68.55 129060 sterol 388, 314 0.700988 0.596422

Chapter 6: Appendix 6.1 514

Appendix 6.1v Cabestana spengleri Fraction 1 R. t. Area Identity M+/ fragments % fraction % total 28.35 17101 unknown 141 0.369176 0.129512 38.117 61555 me ester 111,102 1.328849 0.46618 39.067 16778 unsaturated oxygenated 107, 95 0.362203 0.127066 39.6 14195 unsaturated oxygenated 136, 111 0.306442 0.107504 41.733 114873 acid 73, 60 2.479877 0.869978 43.217 60112 acid 1.297697 0.455251 44.1 49618 acid 1.071153 0.375776 45.85 35227 aldehyde 114 0.76048 0.266788 46.517 126829 acid 2.737983 0.960525 46.767 159652 amide 3.446566 1.209106 50.317 75190 unsaturated oxygenated 1.623201 0.569443 50.467 88514 unsaturated 1.910839 0.670351 50.883 31417 unsaturated 0.67823 0.237933 54.017 56506 ene ol 109, 85 1.219851 0.427942 54.567 38651 unsaturated 0.834397 0.292719 59.5 39102 alcohol 155, 101 0.844134 0.296135 63.433 50554 unsaturated (sterol) 300 1.091359 0.382865 63.75 151967 sterol 385 3.280662 1.150905 64.65 1855614 cholesterol 386 40.05898 14.05328 64.867 146796 ketone 187, 165 3.169031 1.111743 65.25 607757 sterol 384 13.12025 4.602778 65.85 78822 sterol 364, 271 1.701609 0.596949 66.25 146676 sterol 314, 299 3.16644 1.110834 66.367 201436 sterol 400, 289 4.348599 1.525553 66.867 46791 sterol 397, 391 1.010124 0.354366 68.167 189239 sterol 396, 303 4.08529 1.43318 68.483 51056 sterol 314 1.102196 0.386667 69.05 97767 sterol 379 2.110593 0.740427 69.417 22410 sterol 392, 377 0.483787 0.16972

Fraction 2 R. t. Area Identity M+/ fragments % fraction % total 9.533 150854 amine 87, 86, 57 1.759861 1.142476 17.883 69772 unknown 106, 91, 57 0.813959 0.52841 30.817 66095 acid 0.771063 0.500563 43.25 72855 acid 270 0.849925 0.551759 43.45 43718 oxy alcohol 118, 98 0.510014 0.331093 44.15 73341 acid 0.855595 0.55544 44.317 29727 acid 0.346795 0.225134 44.467 169157 unknown 73 1.973383 1.281091 44.633 46085 alcohol 266, 224 0.537627 0.349019 45.167 259953 amine 58 3.032608 1.968724 45.883 269775 amide 98, 85 3.147191 2.04311 45.983 102332 alcohol 97, 73 1.193804 0.775 46.65 279353 acid 3.258928 2.115648 47.067 103689 unknown 98, 85 1.209634 0.785277 47.333 39642 unknown 196, 185 0.462463 0.300224 47.467 41386 unknown 111, 97 0.482808 0.313432 48.183 47835 unknown 98, 85 0.558042 0.362273 Chapter 6: Appendix 6.1 515

Appendix 6.1v Cabestana spengleri Fraction 2 continued. R. t. Area Identity M+/ fragments % fraction % total 48.75 35289 acid 210, 200 0.411681 0.267257 49.117 626282 unknown 73 7.306197 4.743075 49.6 104189 unsaturated 1.215467 0.789063 49.8 41208 unsaturated ketone 98 0.480732 0.312084 50.217 112360 unknown 103 1.31079 0.850946 50.4 189107 unknown 98, 85 2.20612 1.43218 50.533 141820 alkene/ alcohol 111, 97 1.65447 1.074058 50.7 90430 unknown 103 1.054955 0.684861 50.933 59237 unsaturated 0.691058 0.448625 51.6 171708 unknown 103, 57 2.003143 1.300411 52.05 42929 unknown 103, 57 0.500809 0.325118 52.4 56876 unknown 103, 57 0.663515 0.430744 53.017 308844 unknown 125, 98 3.60297 2.338995 53.15 441757 unknown 98, 85 5.153531 3.345596 53.733 60244 unsaturated oxygenated 108 0.702806 0.456251 53.95 54981 unknown 292, 180 0.641408 0.416392 54.5 355472 unknown 103 4.146931 2.692127 54.6 46919 unknown 103 0.547356 0.355336 54.917 328654 unknown 103 3.834073 2.489024 55.467 96293 amide 104, 72, 59 1.123353 0.729264 56.835 63049 unknown 124, 111 0.735529 0.477494 57.167 156897 unknown 152, 98 1.830358 1.188242 57.45 104915 unknown 180, 152 1.223937 0.794562 58.333 242766 polyunsaturated oxygenated 159, 145 2.832105 1.838561 59.15 176239 ene one 366, 253 2.056002 1.334726 59.633 939658 unknown 217, 173 10.96204 7.116392 59.883 68670 unknown 253, 199 0.801103 0.520064 59.983 72589 unknown 368, 314 0.846822 0.549744 60.617 223758 unknown 369, 261 2.610358 1.694606 62.45 110307 unknown 253, 199 1.28684 0.835397 64.333 936501 cholesterol 386 10.92521 7.092483 65.167 246412 sterol 399 2.874639 1.866173

Chapter 6: Appendix 6.1 516

Appendix 6.1w Conuber sordidus 1 Fraction 1 R. t. Area Identity M+/ fragments % fraction % total 6.233 1.8 C8 amine M+ 129 0.538439 0.520833 23.333 3.1 C11 alkene M+ 154 0.927311 0.896991 24.317 0.9 unknown 203, 232 0.269219 0.260417 24.55 1.4 unknown (thiol?) 132, 156, 160 0.418786 0.405093 24.85 1.7 (C12 diene?) M+? 166 0.508525 0.491898 25.15 0.7 unknown 194, 237, 255 0.209393 0.202546 25.733 16.2 unknown 127, 139, 167 4.845947 4.6875 27.817 3.5 C10 alcohol M+ 158 1.046964 1.012731 28.25 1.4 alkane 0.418786 0.405093 28.333 1.4 unknown 141, 162, 191 0.418786 0.405093 29.267 2.3 unsat. cyclic oxy ester? 149, 166, 194 0.688005 0.665509 29.45 0.9 phenol 137, 165, 180 0.269219 0.260417 29.683 2.4 alkane 0.717918 0.694444 30.1 0.7 unknown 163, 181, 196 0.209393 0.202546 30.867 1.4 unknown 91, 179 0.418786 0.405093 31.75 2.3 alkane 0.688005 0.665509 34.45 0.8 alcohol? 0.239306 0.231481 34.583 1.4 alkane 0.418786 0.405093 34.667 3.9 alkane 1.166617 1.128472 35.817 1.8 alkane 0.538439 0.520833 37.233 2 C14 acid ethyl ester M+ 256 0.598265 0.578704 37.85 3.4 aldehyde/alcohol 1.017051 0.983796 39.55 3.6 aldehyde/alcohol 1.076877 1.041667 40.083 1.8 alkane 0.538439 0.520833 40.517 9 aldehyde/alcohol 2.692193 2.604167 40.683 2.8 C16 acid me ester 270 0.837571 0.810185 41.967 0.8 alcohol 0.239306 0.231481 42.133 3.4 aldehyde/alcohol 1.017051 0.983796 42.4 5.2 C16 acid ethyl ester M+ 284 1.555489 1.50463 42.767 2.1 acetic acid ester 0.628178 0.607639 43.083 32.1 C18 aldehyde? M+ 286 ? 9.602154 9.288194 44.55 6.2 alcohol? 1.854622 1.793981 44.8 1.2 acid ethyl ester 0.358959 0.347222 46.633 3.7 unknown 43, 75 1.10679 1.070602 47.117 1.7 C20 acid ethyl ester M+ 312 0.508525 0.491898 47.3 2.1 (unsaturated aldehyde?) 0.628178 0.607639 47.467 60.1 acetic acid ester 17.97786 17.39005 49.133 1.1 alcohol 0.329046 0.318287 49.467 1 alkane 296, 316 0.418786 0.405093 49.867 1.1 poly unsaturated 0.329046 0.318287 51.25 0.7 fatty acid ester 0.209393 0.202546 51.567 2.3 alkane 0.688005 0.665509 51.75 26.2 acetic acid ester 7.837272 7.581019 53.617 2.8 alkane 0.837571 0.810185 55.583 4.1 alkane 1.226443 1.186343 57.5 3.4 alkane 1.017051 0.983796 59.2 2.4 unknown 185, 310 0.717918 0.694444 59.333 2.6 alkane 0.777745 0.752315 59.467 6.1 unknown (poly unsaturated) 1.824708 1.765046 Chapter 6: Appendix 6.1 517

Appendix 6.1w Conuber sordidus 1 Fraction 1 continued. R. t. Area Identity M+/ fragments % fraction % total 61.133 2.9 alkane 0.867484 0.83912 62.983 1.7 alkane 0.508525 0.491898 63.833 4.1 sterol 300, 384 1.226443 1.186343 64.9 36.1 sterol 10.79868 10.4456 64.967 38.8 cholesterol 386 11.60634 11.22685 65.783 3.5 sterol 383, 398 1.046964 1.012731 66.333 1.8 sterol 206, 410, 411 0.538439 0.520833

Fraction 2 R. t. Area Identity M+/ fragments % fraction % total 14.867 0.3 C7 aldehyde M+ 100 2.654867 0.086806 18.633 0.9 C9 aldehyde M+ 142 7.964602 0.260417 27.85 0.5 acid propenyl ester 4.424779 0.144676 30.15 0.4 unknown 196, 237 3.539823 0.115741 30.583 0.5 unknown (dione?) 127, 137 4.424779 0.144676 36.35 1.2 long acid or amine 10.61947 0.347222 41.617 2.1 C16 acid 18.58407 0.607639 43.083 1.1 aldehyde/alcohol 9.734513 0.318287 64.367 3.8 cholesterol 386 33.62832 1.099537 68.033 0.5 sterol 372, 404 4.424779 0.144676

Chapter 6: Appendix 6.1 518

Appendix 6.1w Conuber sordidus 2 Fraction 1 R. t. intensity identity M+/ fragments % fraction % total 10.483 747743 phenol 1.481116 0.909104 19.567 1901477 cyclohexane isocyanate 141 3.766412 2.31181 21.75 187946 methyl napthalene 142, 115 0.37228 0.228504 22.083 507094 alkane 1.004443 0.616523 23.167 330380 alkane 0.654411 0.401675 23.5 169296 unknown 158, 116 0.335339 0.20583 23.85 182970 tetra butyl urea 156, 106 0.362424 0.222454 24.6 400149 unknown 161 0.792608 0.4865 25.517 397986 alkane 0.788324 0.48387 25.883 139318 unknown 0.275959 0.169382 26.283 55026 dioic acid 175, 146 0.108995 0.0669 26.45 552677 benzene diacetyl 162, 147 1.094733 0.671943 26.733 129392 unknown 0.256297 0.157314 27.417 219829 octadieneone tetra methyl 180, 165 0.435433 0.267267 28 747280 unknown 163 1.480199 0.908541 28.05 181531 unknown 166 0.359573 0.220705 28.483 1081273 unknown 2.141766 1.314609 28.767 378549 alkane 220, 205 0.749823 0.460239 29.033 117527 phenol di methyl ethyl 206, 191 0.232795 0.142889 29.45 166027 benzoic acid ethoxyethyl ester 194, 166 0.328863 0.201855 29.517 146753 me ester 0.290686 0.178422 29.8 63039 unknown 155, 127 0.124867 0.076643 30.033 154258 unknown 217, 188 0.305552 0.187546 31.4 1181749 unknown 149 2.340787 1.436767 31.55 56368 benzene acetonitrile dimethoxy 177, 162 0.111653 0.068532 31.667 29499 unknown 181, 148 0.058431 0.035865 32.633 838743 acid 1.661367 1.019741 33.217 192321 alkane 0.380946 0.233823 35.467 951535 me ester 242 1.884784 1.156873 37.383 228717 ethyl ester 256 0.453039 0.278073 38.517 13283664 isopropyl mryistate 228 26.31205 16.15024 40.3 5471541 alkane 10.83793 6.652283 41.867 641302 alkane 1.27028 0.779693 42.6 794109 C16 ethyl ester 284 1.572957 0.965475 43.317 679551 C16 ester 256 1.346043 0.826196 44.817 437670 alcohol 0.866929 0.532118 45.1 291687 C18 eneoic acid me ester 296 0.577768 0.354632 46.633 545791 alcohol 1.081093 0.663571 46.817 462184 alcohol 0.915486 0.561922 47.3 653748 C18 ethyl ester 312 1.294932 0.794825 49.033 357851 alcohol 0.708825 0.435074 51.45 272588 alcohol 147, 129 0.539937 0.331412 63.767 256743 sterol 384 0.508552 0.312147 64.1 723417 sterol 384 1.432932 0.879528 64.917 5597263 cholesterol 386 11.08696 6.805136 65.083 4696352 cholesterol 386 9.302451 5.709811 65.633 1159180 methyl cholestadieneol 398, 384 2.296083 1.409328 66.783 525354 methyl cholesterol 400, 382 1.040612 0.638724 67.25 302226 sterol 394 0.598644 0.367445 Chapter 6: Appendix 6.1 519

Appendix 6.1w Conuber sordidus 2 Fraction 1 contd R. t. intensity identity M+/ fragments % fraction % total 68.6 647212 sterol 396 1.281986 0.786878 68.75 249220 alkane 0.493651 0.303001

Fraction 2 R. t. intensity identity M+/ fragments % fraction % total 10.467 32018 unknown 94 0.100795 0.038927 10.717 396725 amine 73, 61 1.248919 0.482337 13 107943 nitro 103, 86 0.339812 0.131237 18 331223 unknown 126, 57 1.042714 0.4027 19.55 401301 isocyanate cyclohexyl 141 1.263325 0.487901 20.617 186204 caprolactam 113, 84 0.586184 0.226386 21.517 265953 acid 0.837239 0.323345 22.083 292323 amine 114, 72 0.920254 0.355405 22.35 310539 butynan acetyl piperdyl 179, 135 0.977599 0.377552 23.717 128418 dioic acid 103 0.404269 0.15613 24.783 180663 acid 0.56874 0.21965 25.033 175611 unknown 127 0.552836 0.213507 27.717 207113 benzoic acid hyroxy me ester 156, 121 0.652007 0.251807 27.867 76562 acid 147 0.241023 0.093084 28.033 1694529 unknown 163 5.334501 2.060203 30.983 615862 di ethyl toluamide 190, 119 1.938778 0.748763 31.083 532097 unknown 127 1.67508 0.646922 31.233 585601 acid 1.843515 0.711972 33.917 164853 acid 0.518969 0.200428 37.067 1203953 acid 3.790132 1.463763 38.217 139980 ester 102 0.440667 0.170187 38.467 472235 acid 242 1.454606 0.568672 39.567 1012954 acid 3.188853 1.231546 40.983 139120 acid 256 0.43796 0.169142 42.633 4170094 acid 256 13.12776 5.069988 42.8 17009 unknown 278, 263 0.053546 0.020679 43.667 707876 acid 270 2.228445 0.860634 43.833 389798 acid 1.227113 0.473915 44.55 1009333 acid 3.177454 1.227144 46.05 390235 C18 dieneoic acid 280 1.228488 0.474447 46.283 881390 oleic acid 282 2.77468 1.071591 46.417 808428 oleic acid 282 2.54499 0.982884 47.15 3339935 acid 284 10.51436 4.060683 48.95 242265 acid 0.762668 0.294545 49.817 571076 ene yne/ tetraene 1.797789 0.694313 49.95 783269 tetraene 234 2.465788 0.952296 50.183 132836 yne ol 246 0.418177 0.161502 50.45 250334 yne ol 0.78807 0.304355 50.617 310260 eneoic acid 0.976721 0.377213 50.8 479380 eneoic acid 292 1.509123 0.582829 51.15 445630 acid 312 1.402876 0.541796 51.283 1825606 unknown 234, 218 5.747141 2.219566 52.417 91909 unknown 300, 285 0.289336 0.111743 53.733 401521 polyunsaturated 1.264017 0.488168 Chapter 6: Appendix 6.1 520

Fraction 2 contd R. t. intensity identity M+/ fragments % fraction % total 54.633 51389 unknown 258, 257, 103 0.161776 0.062479 55.15 317784 ether 340 1.000407 0.386361 59.017 189515 alcohol 0.596607 0.230412 63.95 412289 sterol 384 1.297916 0.50126 64.85 2495023 cholesterol 386 7.854514 3.033442 65.417 600371 sterol 384 1.890012 0.729929 66.4 83265 sterol 400 0.262124 0.101233 66.6 269424 sterol 400 0.848166 0.327565 67.117 116196 sterol 395, 326 0.365793 0.141271 68.417 328224 sterol 399 1.033273 0.399054 Chapter 6: Appendix 6.1 521

Appendix 6.1w Conuber sordidus 3 Fraction 2 R. t. intensity identity M+/ fragments % fraction % total 21.206 66318 acid 0.666068 0.205159 24.533 86439 acid 0.868154 0.267405 24.9 92247 unknown 127 0.926487 0.285373 30.9 192388 acid 1.932258 0.595166 31.817 52372 unknown 250, 235 0.526001 0.162016 33.733 55121 acid 0.55361 0.170521 35.517 36789 acid 0.369492 0.113809 36.783 664325 acid 228 6.672179 2.055136 38.3 145851 acid 242 1.464861 0.4512 38.483 107954 acid 1.084241 0.333963 39.317 309692 acid 242 3.110406 0.958054 40.917 46829 unknown 213, 196 0.470329 0.144869 41.533 935088 eneoic acid 9.3916 2.892761 42.183 1959315 acid 256 19.67847 6.061279 43.417 230381 acid 270 2.313842 0.7127 44.283 256227 acid 2.573428 0.792656 45.617 80982 eneoic acid 0.813347 0.250524 45.9 259270 dieneoic acid me ester 2.60399 0.80207 46.133 425708 eneoic acid 4.275618 1.316958 46.217 478996 eneoic acid 4.810819 1.481808 46.767 1213081 acid 284 12.18364 3.752752 49.667 425437 ene yne 4.272896 1.316119 49.767 643299 polyunsaturated 6.461004 1.990091 50.333 166487 diol 1.67212 0.515039 50.483 306407 eneal 3.077413 0.947892 50.617 230940 eneoic acid 292 2.319457 0.714429 51 102564 alcohol 1.030106 0.317289 53.6 76686 ene yne 0.770199 0.237234 53.667 84789 yne ol 0.851582 0.262301 53.817 103022 unsaturated 277 1.034706 0.318706 54.117 121638 yne 1.221677 0.376296 Chapter 6: Appendix 6.1 522

Appendix 6.1w Conuber sordidus 3 Fraction 1 R. t. intensity identity M+/ fragments % fraction % total 8.467 69539 pinene 136, 93 0.31088 0.215124 11.082 204933 phellandrene 0.916169 0.633975 25.867 251320 unknown 1.123546 0.777476 26.617 379572 polyunsaturated 1.696907 1.174233 26.983 93484 cubene/ germacrene 204, 161 0.417928 0.289199 27.317 181105 polyunsaturated 0.809644 0.560261 37.983 404583 alcohol/ ene 163 1.80872 1.251606 28.338 129892 germacrene 204, 161 0.580692 0.40183 28.463 311137 unknown 1.390963 0.962524 29.324 671074 tetrahydro napthalene dimethyl methyl ethyl 202, 159 3.00009 2.076015 30.216 512244 elemol 189, 161 2.290027 1.584663 30.95 176512 dimethyltoluamide 191, 190 0.789111 0.546052 31.383 548943 tetrahydro furanmethanol 71, 43 2.454093 1.698194 31.867 1418293 unknown 250, 207 6.340591 4.387589 33.4 133541 napthalene methanol 165 0.597006 0.413118 34.708 318539 ene 1.424054 0.985423 35.455 1876337 me ester 8.388313 5.804581 40.267 906970 C16 eneoic acid 268 4.054681 2.805776 40.855 4153972 me ester 270 18.57066 12.85061 41.824 279053 acid 156 1.247529 0.86327 42.417 139872 me ester 284 0.625309 0.432704 42.55 95010 C16 ethyl ester 284 0.42475 0.29392 43.204 248996 aldehyde 221, 165 1.113156 0.770287 43.324 482586 me ester 284 2.157439 1.492914 43.494 641913 sulfur (S8) 2.869723 1.985803 44.76 162344 alcohol 0.725772 0.502223 44.85 106423 dieneoic acid me ester 0.475772 0.329227 45.043 230217 eneoic acid me ester 1.029203 0.712193 45.179 229893 eneoic acid me ester 282 1.027755 0.71119 45.71 153166 acid 298 0.684741 0.47383 48.513 193135 trieneoic acid me ester 0.863425 0.597477 48.642 240368 polyunsaturated 1.074584 0.743595 49.414 111469 C20 dieneoic me ester 322 0.498331 0.344837 49.599 399163 iodo alkane? 268, 191 1.78449 1.234839 51.496 206242 unknown 245 0.922021 0.638024 51.718 424743 iodo alkane? 340, 177 1.898847 1.313972 52.371 178964 unknown 168, 167 0.800073 0.553638 53.197 131615 ene al/ yne 0.588395 0.40716 53.611 317352 alcohol 1.418747 0.981751 54.519 131317 propeneone (dihydroxy methoxy phenyl) phenyl 270, 193 0.587063 0.406238 54.867 82953 unknown 232, 216 0.370848 0.256621 55.976 72381 unknown 283 0.323585 0.223916 57.001 163345 ether/ene 0.730247 0.505319 58.879 174585 alcohol/ ether 215, 187 0.780496 0.540091 60.15 217102 aldehyde 0.970572 0.67162 61.108 169845 sterol 314, 251 0.759306 0.525427 63.928 179479 sterol 384 0.802375 0.555231 64.731 2118100 cholesterol 368 9.469134 6.552492 65.338 333147 sterol 398, 384 1.48936 1.030614 66.388 116823 sterol 399, 366 0.522266 0.3614 Chapter 6: Appendix 6.1 523

Appendix 6.1w Conuber sordidus 3 Fraction 1 contd. R. t. intensity identity M+/ fragments % fraction % total 66.544 292790 sterol 400 1.308941 0.905767 66.817 103055 sterol 388 0.460716 0.318808 67.057 137149 sterol 397 0.613135 0.42428 68.403 361881 sterol 399 1.617818 1.119504 Chapter 6: Appendix 6.1 524

Appendix 6.1w Conuber sordidus 4 R. t. intensity identity M+/ fragments % total 10.783 396323 unknown 73, 61, 43 1.812981 13.083 301274 unknown 61, 43 1.378179 19.617 266096 isocyanate cyclohexyl 141 1.217257 28.533 149763 unknown 141 0.685091 31 161186 diethyltoluamide 190, 119 0.737346 31.45 160397 unknown 71 0.733736 32.667 118737 acid 200, 183 0.543163 35.5 147795 me ester 0.676089 36.85 374307 acid 228 1.712268 37.433 44669 ethyl ester 101 0.204338 38.383 5761309 acid 228 26.35512 39.4 246072 acid 242 1.125657 40.267 872312 alkane 3.990393 40.883 421497 me ester 1.928139 41.417 183210 unsaturated 0.838095 41.633 3989025 phthalate 18.2478 42.2 1171281 acid 256 5.358028 42.6 122637 ethyl ester 285 0.561003 44.383 307857 acid 1.408293 44.9 80816 alcohol 0.369693 45.933 95714 eneoic acid 0.437844 46.1 224850 dieneoic acid 1.028577 46.833 909767 acid 4.161731 47.05 163607 amide 0.748421 47.333 127358 ethyl ester 0.582599 49.667 725484 alkane 3.318728 49.767 180865 unsaturated 0.827367 51.25 333389 tetraene 234 1.525089 59.7 380024 unknown 1.738421 64.017 389655 sterol 384 1.782478 64.833 2255460 cholesterol 386 10.31761 65.45 448739 sterol 398 2.052758 67.183 89635 sterol 398 0.410036 68.5 259191 sterol 400 1.18567

Chapter 6: Appendix 6.1 525

Appendix 6.1w Conuber sordidus freeze dried R. t. intensity identity M+/ fragments % total 21.6 1493568 amine 20.47278 36.483 55739 acid 228 0.764031 37.933 80931 aldehyde 1.109345 38.15 673487 acid 9.231687 39.65 72427 unknown 0.992778 40.6 284031 aldehyde 3.893297 40.767 49283 me ester 270 0.675537 41.183 47779 unknown 0.654921 41.783 214472 acid 282 2.939832 43.133 171491 aldehyde 2.350678 43.25 41575 me ester 0.569881 44.137 62842 acid 270 0.861394 44.633 158838 aldehyde 2.17724 45.033 54108 eneoic acid me ester 232 0.741674 45.833 184420 dieneoic acid 2.5279 45.933 71192 eneoic acid 0.97585 46.033 55704 oleic acid 0.763551 46.133 70022 oleic acid 0.959812 50.383 42782 yne ol 0.586426 51.283 50985 unknown 0.698867 53.533 69092 polyunsaturated 0.947065 60.267 54032 unknown 0.740633 60.633 101415 sterol 1.390126 61.383 48515 sterol 0.66501 63.833 180494 sterol 2.474085 64.633 2038595 cholesterol 27.94363 65.233 233742 sterol 398 3.203971 66.267 78786 sterol 398 1.079943 66.433 216801 sterol 2.971756 66.933 75020 sterol 1.028321 68.25 263216 sterol 3.60798 Chapter 6: Appendix 6.1 526

Appendix 6.1x Sepioteuthis australis 1 Fraction 1 R. t. Area Identity M+/ fragments % fraction % total 25.817 7456 unknown 194, 171 2.725913 0.208512 28.4 12277 unknown 181, 141 4.488471 0.343334 38.133 10812 acid 3.952867 0.302365 41.7 43848 acid 16.03083 1.226238 43.133 11146 ene one 4.074977 0.311705 46.483 10524 ketone/aldehyde 3.847574 0.294311 60.617 17699 unknown 147, 135 6.470754 0.494964 64.5 159761 cholesterol 386 58.40862 4.467822

Fraction 2 R. t. Area Identity M+/ fragments % fraction % total 10.083 199824 diethylene glycol 45 6.051072 5.58821 30.433 283706 urea 174, 132 8.591187 7.934026 30.883 54767 decanoic acid 172 1.658455 1.531595 36.633 82220 C14 acid 2.489787 2.299337 37.85 69977 alkene 2.119044 1.956953 38.317 99682 acid 243 3.018571 2.787673 39.25 44574 acid 1.34979 1.246541 41.067 65838 thiophene dimethyl ethoxy 172, 116 1.993707 1.841203 41.35 92814 unsaturated acid 2.810594 2.595605 42.333 618683 C16 acid 256 18.73496 17.30188 44.25 44942 C16 acid 1.360934 1.256833 44.5 61993 acid 173, 129 1.877272 1.733675 45.05 60386 phenyl 232, 117 1.828609 1.688734 45.383 78484 amine 58 2.376653 2.194857 46.183 188597 unsaturated acid 5.711096 5.27424 46.9 451043 acid 284 13.65849 12.61372 49.7 126314 polyunsaturated 3.825041 3.532454 53.733 197647 unknown 113, 105 5.985148 5.527329 60.333 76335 unknown 366, 230 2.311577 2.134759 64.567 404465 sterol 387, 368 12.24801 11.31113 Chapter 6: Appendix 6. 527

Appendix 6.1x Sepioteuthis australis 2 Fraction 1 R. t. Area Identity M+/ fragments % fraction 1 % total 25.817 7456 unknown 2.725913 1.362957 28.4 12277 unknown 141 4.488471 2.244235 38.133 10812 acid 3.952867 1.976433 41.7 43848 acid 16.03083 8.015414 43.133 11146 ene one 4.074977 2.037489 46.483 10524 ketone 3.847574 1.923787 66.617 17699 unknown 147 6.470754 3.235377 64.5 159761 cholesterol 386 58.40862 29.20431

Fraction 2 R. t. Area Identity M+/ fragments % fraction 1 % total 10.083 199824 diethylene glycol 7.200893 3.600447 30.433 283706 urea 174 10.22368 5.11184 30.433 54767 decanoic acid 172 1.973593 0.986797 36.633 82220 c14 fatty acid 2.962895 1.481447 37.85 69977 alkene 2.521704 1.260852 38.317 99682 fatty acid 243 3.592158 1.796079 39.25 44574 fatty acid 1.606277 0.803138 41.067 65838 thiophene dimethyl ethoxy 172 2.37255 1.186275 41.35 92814 unsat. Acid 3.344662 1.672331 42.333 618683 C16 fatty acid 256 22.29497 11.14749 44.25 44942 C16 acid 1.619538 0.809769 44.5 61993 acid 173 2.233991 1.116995 45.05 60386 phenyl 117, 232 2.176081 1.08804 45.383 78484 amine 58, 122 2.828263 1.414132 46.183 188597 acid 284 6.796315 3.398158 49.7 126314 unsat. 4.551874 2.275937 53.733 197647 unknown 105, 113 7.122443 3.561221 60.333 76.335 unknown 366 0.002751 0.001375 64.567 404465 cholesterol 14.57537 7.287687 Chapter 6: Appendix 6. 2 528

Appendix 6.2

(See paper copy for Appendix 6.2) Chapter 6: Appendix 6.3 529

Appendix 6.3 Brominated compounds detected in an extract from the freeze-dried egg mass of

Dicathais orbita on the GC/MS in chemical ionization mode.

Retention time Identity MH+ (Br79, Br81 ) 32.2 6-Bromo-2-methoxy-3H-indol-3-one 240, 242

32.4 Unknown brominated indole 230, 232

36.0 Unknown brominated indole 256, 258

37.4 6-Bromoindoxyl 212, 214

38.0 Tyrindoleninone 256, 258

39.2 Unknown brominated indole 253, 255

41.6 6-Bromo-2-methylsulfinyl-3H-indol-3-one 272, 274

42.5 6-bromoisatin 226, 228 Chapter 6: Appendix 6.8 530

Appendix 6.4 to 6.7

(See paper copy for Appendicies 6.4, 6.5, 6.6 & 6.7) Chapter 6: Appendix 6.8 531

Appendix 6.8 Appendix 6.8a Estuarine Mud (Lake Illawarra) Fraction 1 R. t. Area Identity M+/ fragments %fraction % total 4.683 1.9 alcohol C6? 1.556102 0.737578 5.533 8 C6 hydroxy ketone 6.552007 3.10559 10.567 1 benzenalkane 0.819001 0.388199 14.733 1 alkane 0.819001 0.388199 18.45 1.3 alkane 1.064701 0.504658 22.033 0.8 alkane 0.655201 0.310559 23.767 1.8 tetrabutyl urea 156 1.474201 0.698758 26.517 0.2 phenyl 0.1638 0.07764 26.617 0.9 dihydro trimethyl quinoline 158, 173 0.737101 0.349379 26.867 0.6 sulfated 64, 174 0.4914 0.232919 27.9 0.6 unknown 69, 177 0.4914 0.232919 28.85 0.7 sulfur S6 0.573301 0.271739 29.35 0.5 ene one 0.4095 0.194099 34.717 2.4 alkane 1.965602 0.931677 35.05 0.7 hexathiepane 256 0.573301 0.271739 35.367 0.6 acid me ester 0.4914 0.232919 37.5 0.5 alkane 0.4095 0.194099 38.933 0.8 phthalate 0.655201 0.310559 40.167 3 eneoic acid 237 2.457002 1.164596 40.75 3.9 C16 acid me ester 270 3.194103 1.513975 41.4 10.6 phthalate 8.681409 4.114907 42.633 0.5 alkane 0.4095 0.194099 43.117 0.4 aldehyde? 160, 243 0.3276 0.15528 43.417 32.3 sulfur S8 256 26.45373 12.53882 43.533 2 unsaturated/cyclic 91, 105 1.638002 0.776398 43.633 1.4 unsaturated 1.146601 0.543478 43.75 0.4 unsaturated 202, 210 0.3276 0.15528 44.467 0.4 C18? acid me ester 0.3276 0.15528 44.9 0.3 C18 trienoic acid 0.2457 0.11646 45.033 0.6 alkane 0.4914 0.232919 45.1 0.5 unknown 264 0.4095 0.194099 45.217 1.2 phytol - C20 eneol 123 0.982801 0.465839 47.317 0.5 alkane 0.4095 0.194099 47.5 0.4 ene ol acetate? 0.3276 0.15528 49.517 0.9 alkane 0.737101 0.349379 50.15 0.2 phthalate 0.1638 0.07764 51.333 1.6 hexadioic acid dioctyl ester 1.310401 0.621118 51.633 0.9 alkane 0.737101 0.349379 53.667 1 alkane 0.819001 0.388199 54.15 3.8 phthalate 3.112203 1.475155 55.633 1 alkane 0.819001 0.388199 57.567 1 alkane 0.819001 0.388199 57.917 2.5 phthalate 2.047502 0.970497 58.017 0.7 phthalate 0.573301 0.271739 59.4 0.9 alkane 0.737101 0.349379 59.517 1 unsaturated alcohol? 0.819001 0.388199 61.183 1.5 alkane 1.228501 0.582298 61.483 2.1 phthalate 1.719902 0.815217 Chapter 6: Appendix 6.8 532

Appendix 6.8a Estuarine Mud Fraction 1 continued. R. t. Area Identity M+/ fragments %fraction % total 62.333 0.5 alkane? 0.4095 0.194099 62.933 0.6 alkane? 0.4914 0.232919 63.783 2 sterol? 255, 300 1.638002 0.776398 63.967 0.5 alkane? 298 0.4095 0.194099 64.45 3.9 sterol 380 3.194103 1.513975 64.583 0.7 sterol 388 0.573301 0.271739 64.617 0.8 sterol 386 0.655201 0.310559 64.683 0.3 sterol 366 0.2457 0.11646 64.717 0.3 sterol 299 0.2457 0.11646 64.817 0.6 phthalate 0.4914 0.232919 65.133 1.4 sterol 300 1.146601 0.543478 65.433 0.6 sterol 258 0.4914 0.232919 65.717 1.1 sterol 310 0.900901 0.427019 66.333 0.5 sterol 400, 426 0.4095 0.194099 66.617 0.7 sterol 387 0.573301 0.271739 66.9 0.5 sterol 252, 384 0.4095 0.194099 67.667 0.3 sterol 426 0.2457 0.11646 68.133 0.8 sterol 414 0.655201 0.310559 68.2 0.9 sterol 383 0.737101 0.349379 69.367 0.9 sterol 286, 367 0.737101 0.349379 69.7 1.1 sterol 313 0.900901 0.427019 70.067 1.3 sterol 257 1.064701 0.504658

Fraction 2 R. t. Area Identity M+/ fragments %fraction % total 5.55 57.3 C6 hydroxy ketone 42.28782 22.24379 6.983 0.6 dimethyl benzene 106 0.442804 0.232919 7.5 0.6 unknown 140 0.442804 0.232919 9.083 1.1 benzaldehyde 0.811808 0.427019 9.383 3.1 benzene 2.287823 1.203416 9.483 2.2 benzene 1.623616 0.854037 9.967 1.4 benzene 1.03321 0.543478 11.55 1.9 unknown 1.402214 0.737578 30.35 1.2 unknown 217, 218 0.885609 0.465839 30.65 0.4 fatty acid 0.295203 0.15528 36.4 0.5 unknown 0.369004 0.194099 38.417 2.3 oxygenated 1.697417 0.892857 39.067 35.3 unknown 195 26.05166 13.70342 39.533 0.7 aldehyde? 141, 171 0.516605 0.271739 41.183 1.2 eneoic acid 0.885609 0.465839 41.4 6.4 phthalate 4.723247 2.484472 41.717 1.8 C16 acid 256 1.328413 0.698758 51.617 0.6 alkane 0.442804 0.232919 53.65 0.6 alkane 0.442804 0.232919 54.15 1.6 phthalate 1.180812 0.621118 55.633 0.7 alkane 0.516605 0.271739 57.533 0.7 alkane 0.516605 0.271739 57.9 1.3 phthalate 0.95941 0.504658 58 0.4 phthalate 0.295203 0.15528 Chapter 6: Appendix 6.8 533

Fraction 2 contd R. t. Area Identity M+/ fragments %fraction % total 59.383 0.5 alkane 0.369004 0.194099 59.567 8.4 unknown 317 6.199262 3.26087 61.183 0.7 alkane 0.516605 0.271739 61.483 1.2 phthalate 0.885609 0.465839 62.917 0.4 alkane 0.295203 0.15528 64.583 0.4 unknown 395 0.295203 0.15528

Chapter 6: Appendix 6.8 534

Appendix 6.8b Intertidal Pebbles (North Wollongong) Fraction 1 R. t. Area Identity M+/ fragments %fraction % total 3.333 371945 pyridine 17.38189 6.191714 31.8 43706 alkane 2.042487 0.727567 32.467 46273 azobenzene 2.162449 0.7703 34.171 67021 alkane 3.132054 1.115689 36.7 46748 anthracene 152, 178 2.184647 0.778207 37.483 72069 alkane 3.367959 1.199722 38.133 127645 isopropyl myristate 270 5.96516 2.124888 38.95 101427 phthalate 4.739929 1.68844 39.75 15538 methyl phenanthrene 192 0.726128 0.258659 39.883 15873 methyl phenanthrene 192 0.741784 0.264236 40.133 55756 alkane 2.605613 0.928162 41.383 133621 phthalate 6.244433 2.224369 41.65 17568 acid 0.820995 0.292452 41.683 23012 unknown 205, 260 1.075407 0.383077 42.65 39577 alkane 1.849529 0.658833 43.133 15947 unknown 191, 205 0.745242 0.265467 43.4 368359 S8 64, 256 17.21431 6.132019 43.783 68815 fluoranthene 202 3.215892 1.145553 45.017 41372 fluoranthene 202 1.933414 0.688714 47.333 39612 alkane 1.851165 0.659415 49.517 30858 alkane 1.442069 0.513689 51.633 30322 ketone 1.417021 0.504766 53.683 25403 alkane 1.187144 0.42288 54.167 225304 phthalate 10.529 3.750603 57.917 41178 phthalate 1.924348 0.685484 59.567 39550 unknown 91, 101 1.848267 0.658383 61.5 35343 phthalate 1.651664 0.58835

Fraction 2 R. t. Area Identity M+/ fragments %fraction % total 3.333 576065 52, 79 14.8958 9.58967 21.083 21641 acid 0.55959 0.360255 30.4 93157 unknown 132, 174 2.408839 1.550771 30.767 82817 acid 228 2.141469 1.378643 38.15 34422 acid 0.890079 0.573018 39.15 47774 acid 242 1.235332 0.795287 41.167 53713 ene ol 110, 129 1.388902 0.894152 41.4 433051 phthalate 11.19776 7.208937 41.85 465096 acid 12.02638 7.742385 44.133 48828 acid 1.262587 0.812833 45.75 26075 ene ol 109, 121 0.674243 0.434067 45.933 173246 unsaturated 4.479767 2.884001 46.033 97.771 unknown 184, 205 0.002528 0.001628 46.533 198454 acid 5.131592 3.303635 49.517 42429 alkane 1.097122 0.706309 51.617 42639 alkane 218 1.102552 0.709805 53.683 45.867 alkane 0.001186 0.000764 54.183 238421 phthalate 6.165052 3.96896 Chapter 6: Appendix 6.8 535

Appendix 6.8b Intertidal Pebbles, Fraction 2 continued. R. t. Area Identity M+/ fragments %fraction % total 55.633 44704 alkane 1.155949 0.744181 57.55 48146 alkane 1.244952 0.801479 57.933 201218 phthalate 5.203063 3.349647 58.033 52909 phthalate 1.368112 0.880768 59.4 35249 alkane 0.911463 0.586785 59.6 706503 unknown 173, 217 18.26864 11.76105 61.183 63113 alkane 1.631966 1.050633 61.5 137485 phthalate 3.555065 2.288693

Chapter 6: Appendix 6.8 536

Appendix 6.8c Seawater (Towradgi)

R. t. Area Identity M+/ fragments % total 10.317 317522 phenol 1.264177 12.483 192291 unknown 44, 79 0.765584 17.917 492836 oxy alchohol 75 1.962169 19.983 330169 C6 dioic acid di methyl ester 1.314529 31.6 107619 ester 0.428472 32.55 43347 acid 200 0.172581 33.633 1293342 unknown 40, 44 5.14929 31.783 328103 alkane 1.306304 34.833 259830 unknown 105, 123 1.034483 37.5 472340 alkane 1.880566 38.017 441879 dioic acid 143, 157 1.759289 38.15 13069087 isopropyl myristate 228 52.03304 39.65 345394 alkene 1.375146 41.367 2845860 phthalate 11.33046 43.117 293350 unsat. Aldehyde? 110, 124 1.167939 54.133 4283933 phthalate 17.05598

Chapter 6: Appendix 6.8 537

Appendix 6.8d Chloroform control Fraction 1 R.t. Area Identity M+/ fragments %fraction % total 6 773,005 butanone 72 0.88926 0.426934 9.583 90,597 butanamide 129 0.104222 0.050037 12.65 138,999 hexadione 0.159904 0.07677 19.117 114,808 Benzothiazole 135 0.132074 0.063409 21.55 217,223 indol/benzene acetonitrile 117 0.249892 0.119973 26.767 1,015,566 dinitrobenzene 168 1.168301 0.560901 27.267 63,709 unknown 0.07329 0.035187 31.017 47,619 alcohol 0.054781 0.0263 31.267 155,136 phthalate 0.178467 0.085682 31.767 101,286 alkane 0.116519 0.055941 32.4 109,182 tetroxane, benzophenone 182 0.125602 0.060302 32.533 135,783 unknown (acid?) -201 0.156204 0.074993 34.017 225,587 alkene 0.259514 0.124593 34.7 179,924 alkane 0.206983 0.099373 36.85 103,681 Benzene-sulfonamide 213 0.119274 0.057263 36.983 115,151 unknown -180 0.132469 0.063598 37.483 166,416 alkane 0.191444 0.091912 38.183 9,278,183 unknown (acid?) 10.67356 5.124376 39.15 12,272,857 Benzene-dicarboxylic acid 14.11862 6.778346 39.583 232,027 alcohol or alkene 0.266922 0.128149 40.15 295,061 Benzene- dicarboxylic acid 0.339436 0.162963 40.55 55,439 unknown -161 0.063777 0.030619 40.733 184,305 fatty acid me ester 0.212023 0.101792 41.017 59574 unknown -210 0.068534 0.032903 41.533 11,219,388 Benzene-dicarboxylic acid ester 12.90671 6.196511 41.633 156,128 mercaptobenzo-thiazole 167 0.179609 0.08623 42.617 208,232 alkane 0.239549 0.115007 43.333 909,084 sulfur 256 1.045804 0.502091 43.567 106,931 unknown -167 0.123013 0.059058 44.333 88,375 alkane 0.101666 0.04881 44.617 348,764 alcohol,alkene or ethenyloxy 0.401216 0.192624 44.95 109,065 unknown (117) 232 0.125468 0.060237 45.017 358,552 alkane 0.412476 0.19803 45.6 124,722 fatty acid me ester C18? 0.143479 0.068884 46.017 176,754 alkane 0.203337 0.097622 47.317 529,740 alkane 0.609409 0.292577 47.483 428,175 acetic acid ester 0.49257 0.236483 48.9 79,295 alcohol 0.09122 0.043795 49.517 717,157 alkane 0.825013 0.396089 49.817 93,116 alkene, alcohol, aldehyde 0.10712 0.051428 50.167 2,031,723 Benzene dicarboxylic acid ester 334 2.337281 1.122128 50.333 111,075 unknown (211, 268) 0.12778 0.061347 50.433 116,084 alkene 0.133542 0.064114 51.617 1,129,275 alkane 1.299111 0.623703 51.783 174,002 acetic acid ester 0.200171 0.096102 53.55 692,668 Benzene dicarboxylic acid ester 0.796841 0.382563 53.683 1,407,503 alkane 1.619182 0.777369 54.333 10,055,613 Dioctyl phthalate 370 11.56791 5.553754 Chapter 6: Appendix 6.8 538

Fraction 1 contd R.t. Area Identity M+/ fragments %fraction % total 54.917 150,678 alkane/phthalate 0.173339 0.08322 55 118,420 alcohol/phthalate 0.13623 0.065404 55.433 76,348 phthalate 0.08783 0.042167 55.667 1,538,313 alkane 1.769665 0.849616 56.067 187,985 alkane/alcohol 0.216257 0.103825 57.583 1,206,158 alkane 1.387556 0.666166 58.067 10,079,392 Dioctyl phthalate 11.59527 5.566887 59.417 1,246,481 alkane 1.433944 0.688436 59.517 137,834 dodecatrienal 220 0.158563 0.076126 61.2 608,917 alkane 0.700494 0.336307 61.633 10,204,606 Benzene dicarboxylic acid ester 11.73931 5.636043 62.917 660,369 alkane 0.759684 0.364724 64.6 506,962 alkane 0.583206 0.279997 64.85 2,170,605 Benzene dicarboxylic acid ester 2.49705 1.198834 66.383 223,721 alkane 0.257367 0.123562 68.1 307,444 unsaturated ketone 0.353682 0.169803

Fraction 2 R.t. Area Identity M+/ fragments %fraction % total 4.217 418,635 propyl acetate 0.444727 0.231214 6.067 10,614,833 butanone 72 11.27642 5.862613 10.05 2,165,702 ethanol oxy bis 106 2.300684 1.196126 12.683 2,413,095 hexadione 2.563496 1.332762 12.85 199,675 cyclohexenone methyl 110 0.21212 0.110281 12.95 224,122 ethoxy propanol 0.238091 0.123783 15.183 1,056,039 unsaturated ketone 108, 109 1.121859 0.583254 15.717 1,592,155 unsaturated ketone 108, 109 1.691389 0.879353 16.1 789,690 acetic acid ester 0.838909 0.436149 16.4 462,083 furanyl ketone 152, 166 0.490883 0.25521 16.85 253,120 unsat/cyclic ketone 152, 166 0.268896 0.139799 17.717 60,651 unsaturated ketone 43, 101 0.064431 0.033498 17.9 1,227,395 oxy alcohol 1.303895 0.677895 18.067 1,079,583 hydroxy ketone C6 1.14687 0.596258 19.15 457,371 Benzathiozole 135 0.485878 0.252608 19.417 3,677,954 hydroxy ketone 43, 84 3.90719 2.031348 19.65 7,763,417 dione (C8) 8.247287 4.287765 21.25 1,334,621 C6 enediol 43, 83 1.417804 0.737116 21.767 820,562 ene one 43, 84 0.871705 0.4532 21.937 194,731 ketone 114 0.206868 0.107551 22.8 434,451 ethanone dimethyl oxiranyl 43, 99 0.461529 0.239949 23.183 639,171 pentanone propynyloxy 101 0.679009 0.353017 23.767 3,238,073 ene one 3.439892 1.7884 25.55 126,307 acetic acid diethyloxy phosphinyl ethyl ester 179, 197 0.134179 0.06976 25.767 657,331 cycloalkane or amine 58, 125 0.6983 0.363046 25.967 3,257,753 dione 182 3.460799 1.79927 26.217 1,388,304 dione 1.474833 0.766766 26.317 704,506 dienacetate 122 0.748416 0.389101 26.5 587,302 dione 99 0.623907 0.324369 26.95 1,527,997 unknown 109, 121 1.623232 0.843919 Chapter 6: Appendix 6.8 539

Fraction 2 contd R.t. Area Identity M+/ fragments %fraction % total 27.3 2,904,615 dione? 109, 182 3.085651 1.60423 27.55 3,292,292 ene one 43, 82 3.49749 1.818346 28.867 3,619,916 amine 58 3.845534 1.999294 29.633 2,231,805 dione 99 2.370907 1.232635 30.383 859,260 alcohol? 132, 174 0.912815 0.474573 34.1 411,087 thienopyridinone 96, 151 0.436709 0.227045 34.867 3,395,751 unknown 58 3.607398 1.875486 34.967 2,283,794 unknown 58 2.426136 1.261348 35.25 920,766 unknown 71, 152 0.978155 0.508543 35.517 2,829,848 ketone or dione 71, 129 3.006224 1.562936 36.567 341,721 C14 fatty acid 228 0.363019 0.188734 39.15 3,429,123 acetamide ethoxy hydroxy phenyl 153, 195 3.64285 1.893918 39.25 347,383 C15 acid 242 0.369034 0.191861 40.35 1,176,220 amine? 58 1.24953 0.649631 41.4 1,689,811 plastisizer 1.795132 0.933289 41.867 831,582 fatty acid 256 0.883412 0.459286 42.017 1,421,024 fatty acid 256 1.509592 0.784837 45.983 292,808 fatty acid 264 0.311058 0.161719 46.667 953,041 fatty acid 284 1.012441 0.526368 46.917 838,102 amide 72, 284 0.890338 0.462887 50.667 303,341 octadecenamide 72, 281 0.322247 0.167536 51.267 1,086,863 amide 59, 72 1.154604 0.600279 54.167 967,024 plastisizer 1.027296 0.534091 55.633 224,057 alkene 0.238022 0.123748 57.9 743,060 plastisizer 0.789373 0.410395 58.85 218,104 octadecenamide 0.231698 0.12046 59.667 6,100,441 alcohol or oxy 173 6.480663 3.369297 61.483 575,433 plastisizer 0.611298 0.317814 62.9 271,199 alkane 0.288102 0.149784 63.55 206,880 unsat. acid or oxy 394 0.219774 0.114261

Chapter 6: Appendix 6.8 540

Appendix 6.8e Dichloromethane Fraction 1 R. t. Area Identity M+/ fragments %fraction % total 26.567 63779 quinilone 0.19194 0.126905 26.717 163895 dinitrobenzene 168 0.493235 0.326112 27.25 80764 sulphone 164 0.243056 0.160701 27.85 42467 alkene 0.127803 0.084499 31 39836 alhohol or alkene 0.119885 0.079264 31.25 132979 diethyl phthalate 222 0.400195 0.264596 32.383 59699 benzophenone 105, 182 0.179662 0.118787 34 54.319 alcohol 0.000163 0.000108 34.667 60217 alkane 0.181221 0.119817 38.1 143253 acid me ester 102 0.431114 0.285039 39.183 12879817 phthalate 38.76129 25.62774 39.583 46529 alcohol 0.140027 0.092582 40.15 328966 phthalate 0.99001 0.654563 41.5 10509468 phthalate 31.62782 20.91132 42.6 95086 alkane 179 0.286157 0.189198 43.567 79659 unknown 167, 272 0.239731 0.158502 44.583 73849 unsat. oxygenated 0.222246 0.146942 44.933 85007 unknown 215, 232 0.255825 0.169144 45.017 128119 alkane 0.385569 0.254926 47.283 138670 alkane 0.417322 0.27592 47.467 70226 acetic acid ester 0.211342 0.139733 49.483 335811 alkane 1.01061 0.668183 50.117 160067 phthalate 0.481715 0.318495 50.3 56554 unknown 211, 268 0.170197 0.112529 51.3 57666 alcohol 0.173543 0.114741 51.6 457570 alkane 1.377038 0.910454 53.533 708256 phthalate 2.131468 1.409259 53.633 448520 alkane 1.349803 0.892447 54.167 1578050 phthalate 4.749077 3.13994 54.85 72981 dibenxenpyranol 0.219633 0.145215 54.967 55311 phthalate 0.166456 0.110056 55.617 413148 alkane 1.243352 0.822065 57.533 345967 alkane 1.041174 0.688391 57.9 1234148 phthalate 3.714118 2.455658 57.983 439812 phthalate 1.323596 0.87512 59.367 357833 alkane 1.076884 0.712002 59.483 108334 trienenitrite 231 0.326027 0.215559 61.167 270815 alkane 0.815007 0.538857 62.867 241047 alkane 0.725421 0.479626 64.333 71175 unsat. ketone 301 0.214198 0.141621 64.567 195191 alkane 0.587419 0.388383 64.8 227671 phthalate 0.685167 0.453011 66.35 95799 alkane 0.288303 0.190617 71.6 74495 alkane 323 0.22419 0.148227 Chapter 6: Appendix 6.8 541

Fraction 2 R. t. Area Identity M+/ fragments %fraction % total 15.817 13274211 ethanol oxy bis 77.9517 26.41249 18.967 404230 alanine ethyl ester 102 2.373807 0.804321 19.417 135830 acetic acid ethoxy 102 0.79765 0.270269 19.717 647687 pentanone hydroxy methyl 116 3.803488 1.288742 27.383 34878 unknown 152 0.204818 0.069399 30.4 295058 unknown (alcohol) 132, 174 1.732704 0.587095 33.683 65028 thienopyridinone 151 0.381872 0.12939 38.5 158534 acetamide ethoxyhydroxyphenyl 153, 195 0.930978 0.315445 41.317 93956 phthalate 0.551749 0.18695 46.067 42704 oxy 103 0.250776 0.084971 50.317 63597 alcohol 173, 217 0.373468 0.126543 59.55 1813050 ethoxy alcohol 173, 217 10.64699 3.607534

Chapter 6: Appendix 6.8 542

Appendix 6.8f Chloroform control derivatised

R. t. Area Identity M+/ fragments % total 3.65 429693 benzaldehyde 91, 92 0.653142 3.75 2366343 alkane 85 3.596888 3.9 1248242 dimethyl cyclohexane 112 1.897352 4.35 4913309 alkane 7.468326 5.25 304017 nitroalkane 57, 83 0.462112 5.983 1576235 benzene 91, 106 2.395908 6.267 4286815 benzene 6.516043 6.967 1729053 benzene 2.628195 7.267 1914910 alkane 2.910701 10.6 588341 benzene 105, 120 0.89429 10.9 2652899 alkane 4.032459 11.75 1260338 alkane 1.915739 11.783 453522 hexane thiol 0.689362 13.067 603480 alkane 0.917301 13.2 711479 alkane 1.081462 13.383 1502508 alkane 2.283842 13.6 944095 alkane 1.435043 14.75 11188968 alkane 17.00745 15.133 272007 alkyne 0.413456 15.367 650467 alkane 0.988723 15.7 832940 aldehyde 1.266085 15.983 152655 cyclohexane 0.232039 17.15 2981512 alkane 4.531957 17.367 1179745 alkane 1.793236 18.467 11.13364 alkane 1.69E-05 18.917 2579256 alkane 3.92052 20.783 1303994 alkane 1.982096 21.067 3048033 alkane 4.63307 21.75 598315 napthalene 141, 142 0.909451 22.083 8385472 alkane 12.74608 23.6 2147706 triacetin 103, 145 3.264555 25.5 1316122 alkane 2.000531 38.75 698209 alkane 1.061291 31.367 715445 phthalate 1.08749 54.217 252488 phthalate 0.383787 Chapter 6: Appendix 6.8 543

Appendix 6.8g Diethyl ether control

R. t. Area Identity M+/ fragments % total 22.017 950718 alkane 0.17119 24.95 676257 benzene triol 108, 126 0.121769 25.467 2072995 alkane 126 0.373271 26.933 6104331 phthalate 163, 194 1.099168 27.383 8621890 unknown 205, 220 1.552489 27.667 3795282 terpene 203, 218 0.683392 28.75 24718086 BHT 205, 220 4.450828 30.233 11687572 thiophene 181, 196 2.104507 31.367 26601784 phthalate 4.790014 32.5 8882779 benzophenone 182 1.599465 33.367 1082693 terpene 205, 248 0.194954 36.183 1719467 phenol 219, 234 0.309613 41.417 13496625 phthalate 2.430251 50.35 261633 unknown 147, 368 0.047111 54.183 10222590 phthalate 1.840717 58.4 425995475 phenol 203, 219 76.70629 59.333 911923 alkane 183 0.164204 66.417 7557127 phenol 203, 219 1.360764