W.C.Dulap et al.I Methods 4(2007)358-376 359 more than 580 million years ago (the Precambrian)are 2.Bioactive and cytotoxic metabolites from free-living reshwater,estuarine and marine microalgae from oxygen and nutrient exchange to fuel aerobic metab The phytochemistry of microalgal metabolites has olism,since extant forms of symbioses are common to the long been dominated by investigations into the potent toxins produced by harmful phytoplankton blooms nd their endobionts exhibit a high of host gh the ficity and stability that suggests the potential of early trophic food chan,particularly byer-feedingshefish mixtures of biosynthetic congeners.Key representative tances.eates pose serious es pose risks the genera Alexd soning (PSP):okadaic acid (3)and related toxinso Dinophysis and Prorocentrum dinoflagelates are the Man ，of thes shellfish oning (ASP) as (5 and 6) metabolites are potent across a broad spectrum of activi- the dinoflagelate Karenia brevis (formerly Gymnodiniu ties,including antiviral,antibiotic,,and anti rere)cause neurotoxic shellfish poisoning (NSP):cigua cancer d from the notably sponges,tuni Microcystis and Nodularia produce e s been consisten mic ocystins（e.g bioactive metabolites9there is a current surge of inter ates to cause fatal estuarine toxic syn est in the phytochemistry of marine microorganisms for drome. The of harmful microalgal toxins is appreciation largely xtensive and revie 01g toxins was published by Van Dolah [18]and several paper,we exploit the niche idea that marine invertebrates lsrange of m nificance photosynthetic endosymbionts [16].Accordingly, N selecting these phytosymbiotic ass c accumulatior saxitoxin (1) phytoto xins and other we d tion symbioses and host bioaccumulation of phytochemical O H metabolites.We provide some important examples of bio NH Eue Thei Promt Payy H-H search and identify key biosynthetic genes from the cur- 0 ently non-culturable microbial consortia and present e rging technologies or cloning g the biosyntn to achieve a sustainable supply more than 580 million years ago (the Precambrian) are the most primitive extant animals. Early metazoans may have developed with photosymbiotic partners to benefit from oxygen and nutrient exchange to fuel aerobic metab￾olism, since extant forms of symbioses are common to the cyanobacteria–sponge assemblage that persist today. It has been noted that the associations between sponges and their endobionts exhibit a high degree of host speci- ficity and stability that suggests the potential of early coevolution between the host sponge and its microbial symbionts [5,6]. Free-living cyanobacteria have been intensely studied in aquatic environments as the progenitors of harmful substances. In eutrophic waters, red tide blooms of cyano￾bacteria and toxic dinoflagellates pose serious risks to human health from the consumption of seafood contam￾inated by toxic species. In addition to specific phytotox￾ins, it is well recognized that genetically diverse marine and freshwater microalgae, including the prokaryotic cya￾nobacteria, present a valuable resource in the discovery of biomedicinal secondary metabolites. Many of these metabolites are potent across a broad spectrum of activi￾ties, including antiviral, antibiotic, antifungal, and anti￾cancer properties of pharmaceutical interest. The ‘‘drugs-from-the-sea’’ effort spanning the last several dec￾ades has focused primarily on anti-tumor agents sourced from marine sessile invertebrates, notably sponges, tuni￾cates and bryozoans [7,8]. While there has been consistent effort to screen marine photosynthetic microorganisms for bioactive metabolites [9], there is a current surge of inter￾est in the phytochemistry of marine microorganisms for drug discovery [10–12]. This appreciation owes largely to the realisation that many bioactive metabolites origi￾nally attributed to the source animal are actually pro￾duced by their microbial consortia [10,13,14]. In this paper, we exploit the niche idea that marine invertebrates are the ‘‘petri dish’’ that sustains a diverse range of micro￾bial life [15], and that phytochemicals of biomedicinal sig￾nificance can be sourced from animals harbouring photosynthetic endosymbionts [16]. Accordingly, we affirm selecting these phytosymbiotic associations as a selective strategy to optimise performance in the quest to discover novel therapeutics. We present a brief overview of the basic phytochemistry of marine microalgae, including the trophic accumulation of phytotoxins and other bioactive metabolites. We discuss the morphological organisation of microbial-invertebrate symbioses and host bioaccumulation of phytochemical metabolites. We provide some important examples of bio￾active metabolites attributed to marine phytosymbionts and argue their potential role in chemical ecology. Addi￾tionally, we consider the use of molecular techniques to search and identify key biosynthetic genes from the cur￾rently non-culturable microbial consortia and present emerging technologies for cloning the biosynthetic genes for heterologous production of phytochemical metabolites to achieve a sustainable supply. 2. Bioactive and cytotoxic metabolites from free-living freshwater, estuarine and marine microalgae The phytochemistry of microalgal metabolites has long been dominated by investigations into the potent toxins produced by harmful phytoplankton blooms occurring in marine and aquatic environments. Con￾sumption of phytotoxins that accumulate through the trophic food chain, particularly by filter-feeding shellfish, can elicit distinct toxin-specific symptoms of gastrointes￾tinal and neurological illnesses. These phytotoxins are structurally diverse and are often elaborated as complex mixtures of biosynthetic congeners. Key representatives are the saxitoxins (e.g. 1) and gonyautoxins (e.g. 2) from dinoflagellates of the genera Alexandrium, Gymn￾odinium and Pyrodinium causing paralytic shellfish poi￾soning (PSP); okadaic acid (3) and related toxins from Dinophysis and Prorocentrum dinoflagelates are the cause of diarrhetic shellfish poisoning (DSP); domoic acid (4) from Pseudo-nitzschia diatoms cause amnesic shellfish poisoning (ASP); brevetoxins (5 and 6) from the dinoflagelate Karenia brevis (formerly Gymnodinium breve) cause neurotoxic shellfish poisoning (NSP); cigua￾toxin (7) and maitotoxin (8) from the dinoflagellate Gambierdiscus toxicus are the toxic agents of ciguatera fish poisoning (CFP); cyanobacteria of the genera Ana￾baena, Microcystis and Nodularia produce potent hepatotoxins, the microcystins (e.g. 9) and nodularins (e.g. 10); and the elusive toxins of Pfiesteria dinoflagel￾lates are reputed to cause fatal estuarine toxic syn￾drome. The study of harmful microalgal toxins is extensive and has been reviewed elsewhere; an authorita￾tive monograph has been published by UNESCO [17]; a review on the origin and health effects of marine algal toxins was published by Van Dolah [18]; and several overviews on the biochemistry of phytotoxins are avail￾able [9,19–21]. HN NH H N H2N H2N O O H NH2 N OH OH saxitoxin (1) NH N N O O N+ NH NH N+ OH OH O H H S O O O H H H S O O O H gonyautoxin-VIII (2) W.C. Dunlap et al. / Methods 42 (2007) 358–376 359