Forty marine algae of the order Caulerpales were chemically investigated. Virtually all produce toxic secondary metabolites of a unique and unprecedented class. These metabolites are generally Linear terpenoids, but unusual structural features such as aldehydes and bis-enol acetate functional groups make these compounds uruque. The conlpounds are t o n c or deterrent toward microorganisms, sea urchin larvae, and herbivous fishes, and when incorporated into diets at naturally occurring concentrations cause mortality in juvenile conch. Concentrations of bioactive metabolites were found to show httle variation in different plant parts such as blades, shpes and holdfasts. Young growlng tips and reproductive structures contained higher concentrahons than mature plant tissues on a dry weight and ash-free dry weight basis. Chemical variation, both qualitahve and quantitative, was observed in different populations of the same species. In the cases examined, algae growing in areas known to have the highest herbivory produced the greatest concentrations and varieties of secondary metabolites. Based upon these observations and pnor feeding preference data, we conclude that chemical defense IS a major factor In the sumval of marine algae within this order.

The ascidian Atapozoa sp. is common in shallow reef habitats throughout the tropical IndoPacific. Collections from the central Philippine Islands, northern Sulawesi (Indonesia), Palau, Kwajalein. and Ant Atoll (near Ponape) were examined for the presence of secondary metabolites. In most cases, nudibranch molluscs Nembrotha spp. were found feeding on the ascidians. Like many dorid nudibranchs, these appear to obtain their defensive chem~cals through their diet. Both ascidians and nudibranchs contained large quantities of a serles of bipyrrole secondary metabolites previously described as the tambjamines. Concentrations of the different tambjamines and a related, highly bluepigmented tetrapyrrole varied among collections of the ascidians and nudlbranchs Overall, concentrations of tambjamines and the tetrapyrrole were hlgher in the nudibranchs, and were the major components of the mucus exuded by Nembrotha spp. when irritated. Tambjamin? A and the tetrapyrrole were present in the nudibranchs but did not occur in detectable ~imounts in .4tapozoa. The ascidian and nudi. branch extracts and isolated metabolites were tested as feeding deterrents toward a variety of carnivorous fishes in field assays on 2 Guam reefs that differed in the types of fishes that fed during the assays. The crude extract, mixtures of tambjamines, tambjamine C, tarnbjamine F, and the tetrapyrrole were all significant feeding deterrents at or below natural concentrations. Tambjamines A and E were not deterrent when tested alone at natural concentrations; however, a I : 1 mixture of talnbjamines E and F was deterrent when tested below natural concentrations. Evidence is thus presented that both the ascidian and its nudibranch predators use the tambjarnines as chemical defenses agalnst predators.

Dorid nudibranchs prey upon sponges, tunicates, and bryozoans which generally contain high levels of secondary metabolites. On Guam, the nudibranch Glossodoris pallida was found in Apra Harbor exclusively on a variety of Hyrt~os erecta sponge containing a high concentration of the secondary metabollte scalaradial. Glossodoris hjkeurensis and G. cincta preyed upon a second variety of H. erecta from Cocos Lagoon which did not contaln scalaradial, but rather had high concentrations of heteronemin, a secondary metabolite that was not found in the variety of H. erecta from Apra Harbor. In laboratory tests, G. pallida strongly preferred H. erecta from Apra Harbor while G. hikeurensis and G. cincta preferred H erecta from Cocos Lagoon. H. erecta from Apra Harbor contained 3 major secondary rnetabolites, scalaradial, deoxyscalarin, and scalarin, as well as several minor metabolites. G. pallida sequestered only 2 of these metabolites, scalaradial and deoxyscalarin. H. erecta from Cocos Lagoon contained primarily heteronemin, whereas its predators G. l~ikeurensis and G. cincta contained heteronemin and episcalaradial. Results of feeding deterrence assays with extracts and isolated metabolites differed when tested toward different predators, indicating that these secondary metabolites may be deterrent toward some but not all potential predators Organlc extracts of the Hyrtios sponges were deterrent at half of natural concentrations in some laboratory and field feeding assays while organic extracts of nudibranchs were never deterrent at half of natural concentrations. Heteronemin deterred feeding by fishes in most assays; however, scalaradial did not deter feeding by predatory fishes in most fleld or laboratory assays and was only deterrent at high concentrations in one assay. Minor metabolites or synergistic effects among sponge metabolltes may play a key role in determining feeding deterrence for these sponges. Glossodoris nudibranchs probably excrete or alter some secondary metabolites from their diets.

We tested the susceptibility to herbivory of 7 species of tropical brown algae from Bundegi Reef in Exmouth Gulf in Western Australia, and also analyzed these algae for the presence of polyphenolics and other non-polar secondary metabolites. All algae contained very low levels of polyphenolics, consistent with findings from other tropical regions, and the susceptibility of the algae to herbivory was not correlated with the variation in phenolic levels that did exist. Levels of polyphenolics In all these algae were probably too low to have any effect on the herbivores, and polyphenolics may in general play little role in defending tropical algae against herbivores. Five algal species, including the 2 species least favored in our grazing assays, contained other, more lipophilic metabolites. Organic extracts from these 2 species deterred feeding by herbivorous fishes; extracts from other species did not. Our data and previous work suggest that unpalatable tropical brown alyde will generally contain low levels of polyphenolics but will contain deterrent, non-polar secondary compounds. The consistently low levels of polyphenolics in tropical brown algae (relative to temperate species) remains a paradox, but may in part be caused by a paucity of trace metals which act as cofactors for biosynthetic systems.

Seaweed preferences of the tropical herbivorous surgeonfish Zebrasorna flavescens were examined in aquarium assays on Guam, in order to examine the role of secondary metabolites in determining its preferences. Of 55 species of macrophytes examined (49 algae, 5 cyanobactena, l seagrass), only 7 species were highly preferred. Seaweed extracts were examined by thin-layer chromatography (TLC) and proton nuclear magnetic resonance (NMR) spectroscopy to determine the presence or absence of secondary metabolites. Extracts from the non-preferred seaweeds and several pure major metabolites from these seaweeds were tested as feeding deterrents by applying them to the surface of Enterornorpha clathrata, a preferred alga. Extracts that contained secondary metabolites and were the most deterrent were from Bryopsis pennata, Chlorodesmis fastigiata, Desmia hornernanni, Microcoleus lyngbyaceus, Liagora farinosa, and Dictyota bartayresii. The deterrent pure metabolites were malygamide A (from M. lyngbyaceus), chlorodesmin (from C. fastigiata), pachydictyol A (from D. bartayresii), ochtodene (from D. hornernanni), and a fatty acid metabolite isolated from L. farinosa. Several extracts which did not contain metabolites based on our chemical analyses were also significantly deterrent including those from Codiurn geppii, Padina tenuis, Galaxaura oblongata, and Halyrnenia durvlllaei. Extracts from Caulerpa racernosa, Avrainvillea obscura, and Asparagopsis taxiformis contained secondary metabolites yet were non-deterrent. The pure metabolites caulerpin, caulerpenyne, and avrainvilleol from C, racernosa and A. obscura were also non-deterrent. Secondary metabolites appear to account for the low palatabhty of many of the seaweeds examined. However, not all secondary metabolites were effective deterrents toward this herbivore.