THE ECOLOGY OF CHEMICAL DEFENCE IN A FILAMENTOUS MARINE RED ALGA NICHOLAS A. PAUL A thesis submitted to the University of New South Wales for the degree of Doctor of Philosophy July 2006 i ACKNOWLEDGEMENTS··························································································································· iii CHAPTER 1. GENERAL INTRODUCTION ..........................................................................1 NATURAL PRODUCTS CHEMISTRY OF MACROALGAE ...............................................................2 THE RED ALGAE ........................................................................................................................2 ECOLOGICAL ROLES OF SECONDARY METABOLITES ................................................................3 i) Chemical mediation of interactions with herbivores ........................................................3 ii) Chemical mediation of interactions with fouling organisms ............................................4 ULTRASTRUCTURE OF SPECIALISED CELLS AND STRUCTURES.................................................5 RESOURCE ALLOCATION TO SECONDARY METABOLITE PRODUCTION.....................................6 THESIS AIMS ..............................................................................................................................7 CHAPTER 2. CHEMICAL DEFENCE AGAINST BACTERIA IN ASPARAGOPSIS ARMATA: LINKING STRUCTURE WITH FUNCTION.......................................................9 INTRODUCTION ..........................................................................................................................9 MATERIAL AND METHODS.......................................................................................................11 Study organism....................................................................................................................11 Screening of algal extracts against known microorganisms ...............................................12 Chemical analysis................................................................................................................13 Quantification of metabolite release ...................................................................................15 Screening of metabolites against known microorganisms ..................................................16 Effect of removing bromine on algal structure and chemistry ............................................16 Bacterial densities on algae cultured in bromide (+) and bromide (-) media. ...................17 Antibacterial assays using bromide (+) and bromide (-) algae ..........................................18 Statistical analysis...............................................................................................................19 RESULTS...................................................................................................................................20 Antibiotic tests of extracts ...................................................................................................20 Natural product chemistry...................................................................................................20 Release of major metabolites ..............................................................................................21 Antibiotic testing of major metabolites................................................................................22 Effect of bromine on cell structure and chemistry...............................................................23 Gland cells and related structures ......................................................................................23 Epiphytic bacterial densities on bromide (+) and bromide (-) algae..................................24 Antibacterial assays with bromide (+) and bromide (-) algae............................................24 DISCUSSION .............................................................................................................................37 Conclusions .........................................................................................................................41 CHAPTER 3. ULTRASTRUCTURE OF THE GLAND CELLS OF THE RED ALGA ASPARAGOPSIS ARMATA (BONNEMAISONIACEAE).....................................................43 INTRODUCTION ........................................................................................................................43 MATERIALS AND METHODS.....................................................................................................45 Study organism....................................................................................................................45 Light Microscopy.................................................................................................................46 Manipulation of gland cells.................................................................................................46 Epifluorescence microscopy................................................................................................47 TEM of cryofixed and freeze-substituted material ..............................................................47 RESULTS...................................................................................................................................48 Light microscopy .................................................................................................................48 Bromine manipulation.........................................................................................................49 Epifluorescence microscopy................................................................................................50 Transmission electron microscopy......................................................................................50 DISCUSSION .............................................................................................................................59 ii CHAPTER 4. SEAWEED-HERBIVORE INTERACTIONS AT SMALL SCALES: A DIRECT TEST OF FEEDING DETERRENCE USING FILAMENTOUS ALGAE .........64 INTRODUCTION ........................................................................................................................64 MATERIALS AND METHODS .....................................................................................................67 Feeding assay with an amphipod ........................................................................................67 Collection and culturing of Asparagopsis armata ..............................................................68 Effects of bromide (+) and bromide (-) artificial media on algae.......................................68 Herbivores...........................................................................................................................69 Feeding assays - Whole plants ............................................................................................70 Feeding assays - Artificial diets ..........................................................................................72 Statistical analysis...............................................................................................................75 RESULTS...................................................................................................................................76 Feeding assays with the amphipod Hyale nigra..................................................................76 Effects of bromide (+) and bromide (-) artificial media on algal thalli..............................76 Whole-plant feeding assays.................................................................................................77 Artificial-diet feeding assays ...............................................................................................78 DISCUSSION .............................................................................................................................88 CHAPTER 5. TRADE-OFFS BETWEEN GROWTH AND CHEMICAL DEFENCE IN THE RED ALGA ASPARAGOPSIS ARMATA .......................................................................95 INTRODUCTION ........................................................................................................................95 METHODS.................................................................................................................................98 Study organism....................................................................................................................98 Chemical analyses...............................................................................................................99 Growth and chemistry across a light gradient....................................................................99 Correlations between growth and metabolite production in the laboratory.....................100 Use of gland cell size to inform allocation to defence.......................................................101 Resource allocation at the cellular scale in the laboratory ..............................................102 Comparison of laboratory-cultured and field-sampled algae...........................................103 Cellular correlates between defence and growth in the field............................................104 Statistical analysis.............................................................................................................105 RESULTS.................................................................................................................................106