Sequestration of dietary secondary metabolites by three species of sea hares: location,specificity and dynamics

We examined the location of squuestered secondary metabolites in three species of sea hares, Stylocheilus longicauda, Dolabella auricularia, and Aplysia californica (Opisthobranchia: Anaspidea). The sea hares ate a natural diet or were fed an artificial diet containing secondary metabolites in the laboratory. In all three species, sequestered secondary metabolites were located almost exclusively in the digestive gland, an internal organ, rather than in the exterior parts of the body, in eggs, or in ink (released when sea hares are disturbed). S. longicauda, a specialist sea hare, was able to sequester measur-able amounts of all six algal metabolites offered (caulerpenyne, halimedatetraacetate, pachydictyol A, malyingamides A and B, and ochtodene) and two (luffariellolide and Dysidea spp. brominated diphenyl ether) of three sponge metabolites offered (chondrillin was not sequestered). Malyngamides A and B, found in the host plant of S. longicauda, were sequestered at high, but not unique concentrations. D. auricularia, a generalist sea hare, was fed caulerpenyne, pachydictyol A and malyngamide B; patterns of sequestration of these three compounds did not differ markedly between S. longicauda and D. auricularia. S. longicauda did not lose measurable amounts of malyngamides after 18 d on a malyngamide-free diet. These results suggest that sea hares have generic mechanisms for sequestering algal metabolites rather than mechanisms that are tightly linked to particular compounds, that these mechanisms do not differ dramatically between species, and that sequestered secondary metabolites are not located optimally for defense.     

A brominated secondary metabolite synthesized by the cyanobacterial symbiont of a marine sponge and accumulation of the crystalline metabolite in the sponge tissue

The dictyoceratid marine sponge Dysidea herbacea (Keller, 1889) is common in shallow waters of the tropical Pacific Ocean. Polybrominated biphenyl ethers such as 2-(2,4-dibromophenyl)-4,6-dibromophenol (1) are characteristic secondary metabolites of some specimens of this sponge and may represent as much as 12% of the dry weight. We have found 1 to be deposited as conspicuous crystals throughout the sponge tissue. The dominant prokaryotic endosymbiont in the mesohyl of the sponge is a filamentous cyanobacterium (Oscillatoria spongeliae), although a vacuole-containing, heterotrophic bacterium is also present. The cyanobacteria were separated from the sponge cells and heterotrophic bacteria by flow cytometry. Coupled gas chromatography—mass spectrometry and proton nuclear magnetic-resonance spectroscopy revealed that the major brominated Compound 1 isolated from the intact symbiotic association is found in the cyanobacteria and not in the sponge cells or heterotrophic bacteria. This suggests that the production of the compound is due to the cyanobacterium, and not to the sponge or symbiotic heterotrophic bacteria, as had been suggested earlier.     

Unravelling host and symbiont phylogenies of halichondrid sponges (Demospongiae, Porifera) using a mitochondrial marker

We present the first comparative phylogenetic analysis of a selected set of marine sponges and their bacterial associates. The Halichondrida form an important order in demosponge systematics and are of a particular interest due to the production of secondary metabolites. We sequenced a fragment of the cytochrome oxidase subunit 1 (CO1) gene of the sponges and their bacterial associates, compared the reconstructed phylogenies and found evidence for radiation in coevolution. The tree of six host-species associations showed four supported cospeciation events between the sponges and the bacteria. In addition, we present the first gene tree of sponges based on a mitochondrial marker. The tree shows major congruences with previous morphological studies and suggests the applicability of a mitochondrial marker in sponge molecular systematics.     

Investigation of the relationship between invertebrate predation and biochemical composition,energy content,spicule armament and toxicity of benthic sponges at McMurdo Sound,Antarctica

The biochemical and energetic composition, spicule content, and toxicity of benthic sponges was investigated in McMurdo Sound, Antarctica from October through December 1984. The predominant organic constituent of sponges was soluble and insoluble protein. Levels of total protein ranged from 17.0 to 55.9% dr. wt. Levels of lipid and carbohydrate were low, ranging from 2.1 to 9.6 and 0.6 to 3.5% dr. wt, respectively. Levels of ash were high and variable (32 t0 79% dr. wt), reflecting species-specific differences in spicule contents. Calculated energy contents of sponges were low, with a mean of 9.8±3.5 kJ g^-1 dr. wt; ranging from 5.1 kJ g^-1 dr. wt in Sphaerotylus antarcticus to 17.4 kJ g^-1 dr. wt in Dendrilla membranosa. Insoluble protein accounted for the greatest contribution to the energetic composition of the sponges, while lipid and carbohydrate combined contributed to less than 25% of the overall energy. Normalized spicule volumes of sponges ranged from 0.15 to 0.38 cm³ g^-1 dr. wt. Ichthyotoxicity assays indicated that 9 (56%) of 16 antarctic sponge species were toxic. The most highly toxic species were Mycale acerata and Leucetta leptorhapsis. The high incidence of toxicity in antarctic sponges indicates that the current hypothesis suggesting a simple inverse relationship between toxicity and latitude in marine sponges is invalid. There was little correspondence between the energetic composition or spicule contents of the sponges and feeding patterns (electivity indices) of sponge-eating predators. Although the asteroid Perknaster fuscus antarcticus specializes on the highly toxic, fast-growing M. acerata, most antarctic sponge-eating predators appear to be generalists which feed on the more abundant, non- to mildly-toxic, sponge species. This feeding strategy is based on exploitation of low energy, sedentary prey, which require a minimal energy output to harvest.     

Sponge-feeding by the Caribbean starfish Oreaster reticulatus

The common Caribbean starfish Oreaster reticulatus (Linnaeus) feeds on sponges by everting its stomach onto a sponge and digesting the tissue, leaving behind the sponge skeleton. In the San Blas Islands, Republic of Panama, 54.2% of the 1549 starfish examined from February 1987 to June 1990 at eight sites were feeding, and 61.4% of these were feeding on sponges, representing 51 species. Sponges were fed on disproportionately heavily in comparison to their abundance, which was only 9.7% of available prey. In feeding choice experiments, 736 pieces of 34 species of common sponges from a variety of shallow-water habitats, and also 9 ind of a coral, were offered to starfish in individual underwater cages. Acceptance or rejection of sponge species was unambiguous for 31 of the 34 species, and there was a clear relationship between sponge acceptability and sponge habitat. Starfish ate 16 of 20 species that normally grow only on the reefs, but only 1 of 14 species that live in the seagrass meadows and rubble flats surrounding the reefs. The starfish live in the seagrass meadows and rubble flats, and avoid the reefs, and so the acceptable reef sponges are generally inaccessible until a storm fragments and transports them into starfish habitat. After Huricane Joan washed fragments of reef sponges into a seagrass meadow in October 1988, starfish consumed the edible species. When the seagrass meadow was experimentally seeded with tagged reef sponge fragments in June 1994, O. reticulatus consumed edible species and accumulated in the area seeded. Reef sponges that were living in a seagrass meadow, from which O. reticulatus had been absent for at least 4 yr (from 1978 to 1982), were eliminated when the starfish migrated into the area, and the sponges have been unable to recolonize up to June 1994. O. reticulatus feeding and habitat preferences appear to restrict distributions of many Caribbean reef sponge species to habitats without O. reticulatus and may have exerted significant selective pressure on defences of those sponges that live in O. reticulatus habitats.     

Distribution of secondary metabolites in the sponge Oceanapia sp. and its ecological implications

The Micronesian sponge Oceanapia sp. has an unusual growth form that consists of an irregular turnip-shaped base, which is buried in the substrate. One to several fistules, which protrude through the sand, are attached to the base of the sponge. On top of each fistule is a small fragile capitum. We examined whether this conspicuous red-colored sponge was chemically defended and if intraspecimen variation existed in the distribution of secondary metabolites between different parts of the sponge. Furthermore we assessed the deterrent properties of the secondary metabolites to generalist and more specialized fish predators. We also wanted to see if the optimal defense theory holds in the case of a marine invertebrate. According to the theory, organisms evolve and allocate defenses in a way that maximizes individual fitness, assuming that defenses are costly to the fitness of the organisms. We were able to evaluate this hypothesis, since the different sponge parts in Oceanapia sp. were at different risk to damage by predators and had a different value in terms of fitness loss to the sponge (the capitum probably plays a role in asexual propagation). Concentrations of crude organic extract increased from the base to the capitum of the sponge. The major secondary metabolites kuanoniamine C and D also showed a sharp increase from the basal root to the capitum. There was no difference in structural material or ash content between the base and the fistule of the sponge, but fiber and protein content were significantly higher in the fistule. The methanol fraction was highly deterrent in field feeding assays towards generalist reef fish at base concentration. It also deterred feeding by the spongivorous angelfish Pomacanthus imperator in laboratory feeding experiments at the same concentration. The field feeding assays with pure compounds showed that kuanoniamine C and D deterred feeding by natural assemblages of reef fishes at fistule concentrations, confirming their role as defensive agents. The intraspecimen variation of secondary metabolites in Oceanapia sp. supports the optimal defense theory by showing the highest concentrations in those parts of the sponge that are most visible to predators and are likely to be most important for inclusive fitness. Received: 5 May 1999 / Accepted: 16 September 1999     

Host specificity in sponge-encrusting zoanthidea (Anthozoa: Zoantharia) of Barbados,West Indies

Six species of common Caribbean Zoanthidea, Parazoanthus swiftii, P. parasiticus, P. catenularis, P. puertoricense, Epizoanthus cutressi, and Epizoanthus sp., are virtually restricted to living on surfaces of reef-dwelling sponges. Quantitative surveys on Barbados reefs indicate that substrate specificity is relatively high among these zoanthids with three restricted to a single primary host sponge species and three restricted to three closely related sponges. One species, P. swiftii, exhibits a broad range of acceptable secondary substrates, due to its unique ability to execute migrational spread in the adult polyp stage. Variations in substrate specificity have been noted between island populations within the extensive Caribbean range and appear to be due to different species compositions of local sponge communities and slight differences in zoanthid larval settling specificities.     

Microbial associations in sponges. II. Numerical analysis of sponge and water bacterial populations

Three taxonomically distant sponges Pericharax heteroraphis, Jaspis stellifera and Neofibularia irata contain phenotypically similar bacterial symbionts which differ from bacteria in the ambient water. These symbionts are predominant in the sponges and were detected after computer analysis of 526 heterotrophic bacterial strains tested for 76 characters. These facultative anaerobic symbionts metabolize a wide range of compounds and may be important in removing waste products while the sponges are not circulating water. The bacteria produce sticky-mucoid colonies and thus would contribute to sponge structural rigidity. The fourth sponge Ircinia wistarii contains a mixed aerobic population similar to that in the ambient water. The majority of the bacteria are located around the inhalant canals, facilitating the uptake of dissolved organic matter and oxygen from the incoming water.     

Sponge-specific bacterial symbionts in the Caribbean sponge, Chondrilla nucula (Demospongiae, Chondrosida)

Marine sponges harbor dense and highly diverse bacterial communities, and some percentage of the microflora appears to be specialized for the sponge habitat. Bacterial diversity was examined in Chondrilla nucula Schmidt to test the hypothesis that some subset of sponge symbiont communities is highly similar regardless of the species of host or habitat requirements of the host. C. nucula was collected from a mangrove channel on Lower Matcumbe Key in the Florida Keys (25°53′N; 80°42′W) in August 1999. Domain-specific universal bacterial primers were used to amplify the 16S rDNA gene from genomic DNA that had been extracted from sponges and the surrounding water. An RFLP technique was used to assess diversity of sponge-associated and environmental bacterial communities. The clone library from C. nucula contained 21 operational taxonomic units (OTUs). None of the 53 OTUs from adjacent water samples were found in the C. nucula library indicating that a distinct community was present in the sponge. Sequence analysis indicated that C. nucula harbors a microbial community as diverse as the microbes from other sponges in different habitats around the world. Phylogenetic analysis placed several C. nucula clones in clades dominated by bacteria that appear to be sponge specialists (e.g., Acidobacteria, Bacteroidetes, and Cyanobacteria). Proportional representation of major bacterial taxonomic groups represented in symbiont communities was compared as a function of geographic location of sponge hosts. This study supports the hypothesis that sponges from different oceans existing in dissimilar habitats harbor closely related bacteria that are distinct from other bacterial lineages and appear specialized for residing within sponges.     

Biodiversity and rockfish recruitment in sponge gardens and bioherms of southern British Columbia,Canada

In the Strait of Georgia and Howe Sound, British Columbia, colonies of individual cloud sponges, growing on rock (known as sponge gardens) receive resource subsidies from the high biodiversity of epifauna on adjacent rock habitats. Bioherms are reefs of glass sponges living on layers of dead sponges. In the same area as the sponge gardens, newly discovered bioherms in Howe Sound, BC (49.34.67 N, 123.16.26 W) at depths of 28- to 35-m are constructed exclusively by Aphrocallistes vastus, the cloud sponge. The sponge gardens had much higher taxon richness than the bioherms. The sponge garden had 106 species from 10 phyla, whereas the bioherm had only 15 species from 5 phyla. For recruiting juvenile rockfish (quillback, Sebastes maliger), the food subsidy of sponge gardens appears to be missing on bioherms of cloud sponge, where biodiversity is relatively low. While adult and subadult rockfishes (S. maliger, S. ruberrimus, S. proriger, and S. elongatus) were present on bioherms, no evidence for nursery recruitment of inshore rockfishes to bioherms was observed, whereas the sponge gardens supported high densities of newly recruited S. maliger, perhaps owing to the combination of both refuge and feeding opportunities. These results indicate that sponge gardens form a habitat for early stages of inshore S. maliger, whereas A. vastus bioherms are associated only with older juvenile and adult rockfishes.     

Trends in space occupation by the encrusting sponge Crambe crambe: variation in shape as a function of size and environment

The relationship between sponge size, habitat and shape was studied in the encrusting sponge Crambe crambe (Schmidt, 1862), which is distributed widely throughout the shallow Mediterranean littoral. Examination of sponge patches in shaded and well-illuminated habitats showed that the degree of peripheral irregularity of the edges of a patch is directly related to patch size. This relationship is valid only for sponges of >100 mm² in area. Photophilic and sciaphilous sponges display different growth forms. The pattern of growth is interpreted in terms of competition for space. The directional growth of sciaphilous sponges may be due to the presence of dominant neighbours that are good space competitors, and the irregular growth of photophilic sponges to the absence of such neighbours.     

Evidence of nitrification and denitrification in high and low microbial abundance sponges

Aerobic and anaerobic microbial key processes were quantified and compared to microbial numbers and morphological structure in Mediterranean sponges. Direct counts on histological sections stained with DAPI showed that sponges with high microbial abundances (HMA sponges) have a denser morphological structure with a reduced aquiferous system compared to low microbial abundance (LMA) sponges. In Dysidea avara, the LMA sponge, rates of nitrification and denitrification were higher than in the HMA sponge Chondrosia reniformis, while anaerobic ammonium oxidation and sulfate reduction were below detection in both species. This study shows that LMA sponges may host physiologically similar microbes with comparable or even higher metabolic rates than HMA sponges, and that anaerobic processes such as denitrification can be found both in HMA and LMA sponges. A higher concentration of microorganisms in the mesohyl of HMA compared to LMA sponges may indicate a stronger retention of and, hence, a possible benefit from associated microbes.     

Cellular localisation of secondary metabolites isolated from the Caribbean sponge <Emphasis Type="Italic">Plakortis simplex</Emphasis>

The Caribbean sponge, Plakortis simplex, is known to contain a large array of secondary metabolites, including the antimalarial polyketide plakortin, several unusual glycolipids, and some hopanoids, which closely resemble typical bacterial metabolites. The hypothesis that they could be products of bacterial metabolism was tested by localizing specific metabolites in cells using physical separation of sponge cells, bacterial symbionts and supernatant by differential centrifugation. The obtained fractions were analysed separately for the typical P. simplex metabolites by NMR and mass spectrometry, and most of them were shown to be present in the bacterial cells but not in the sponge cells. In addition, PCR screening showed that the biosynthetic pathway for glycosphingolipids was present in the bacterial cells. Isolation of a Sphingomonas strain PS193 from P. simplex and subsequent glycosphingolipid analysis resulted in the detection of a known glycosphingolipid, GSL-1, that did, however, not match the glycosphingolipid profile of P. simplex. Therefore, it is unlikely that Sphingomonas strain PS193 is an abundant member of the microbial community associated with P. simplex. Other glycosphingolipid producing bacteria in P. simplex remain to be identified. In conclusion, this study provides experimental evidence that the glycolipids and hopanoids and possibly also the polyketide plakortin are produced by microbial symbionts rather than the sponge from which the metabolites were originally isolated.     

Sponge-feeding fishes of the West Indies

In an analysis of the stomach contents of 212 species of West Indian reef and inshore fishes, sponge remains were found in 21 species. In eleven of these, sponges comprised 6% or more of the stomach contents; it is assumed that these fishes feed intentionally on sponges. Sponges comprise over 95% of the food of angelfishes of the genus Holacanthus, over 70% of the food of species of the related genus Pomacanthus, and more than 85% of the food of the filefish, Cantherhines macrocerus. Lesser quantities of sponges are ingested by the remaining fish species. Fishes that feed on sponges belong to highly specialized teleost families, suggesting that this habit has evolved in geologically late time. The small number of fish species that concentrate on sponges as food suggests that the defensive characters of sponges—mineralized sclerites, noxious chemical substances, and tough fibrous components—are highly effective in discouraging predation. The two sponges most frequently eaten by fishes have a low percentage of siliceous spicules relative to organic matter, but among the 20 next most frequently consumed species no striking correlation occurs with respect to spicule content. Color and form of the sponge show no special correlation with frequency of occurrence in fish stomachs. Three species of fishes appear to concentrate on one species of sponge, but in these cases over 60% of the food taken consists of a variety of other organisms. Those fishes, more than half of whose diet consists of sponges, tend to sample a wide variety of species. No strong evidence is provided by our data that fish predation is a significant factor in limiting sponge distribution in the West Indian region.     

Parrotfish predation on cryptic sponges of Caribbean coral reefs

Some sponge species that live in crevices in the reef frame appear to be restricted to their cryptic habitat by predation. When cryptic sponges were excavated, on Guigalotupo reef, San Blas, Panama, exposing them to potential predators, they were eaten by fishes that are generally considered to be herbivores, primarily parrotfishes of the genus Sparisoma: S.aurofrenatum (Cuvier & Valenciennes), S.viride (Bonnaterre), and S.chrysopterum (Bloch & Schneider). Of the 9150 bites observed to be taken by these species during paired (i.e., with sponges versus without sponges) trials conducted in defined feeding areas during 1986, 1987, and 1988, 72% (i.e. 6581 bites) were on cryptic sponges, even though these were only offered during half of the total observation time and never constituted more than 7% of the cover of the feeding observation areas. Individual parrotfish returned over and over to take bites of the exposed cryptic sponges until they were entirely consumed. They vigorously chased each other away from the sponges, but exhibited no such defense of their usual algal foods. A total of 18 sponge species were tested. Of the cryptic and semi-cryptic sponge species tested, only one of six was rejected by the parrotfish. Two of these six sponge species were consistently consumed entirely, and two were consumed entirely whenever their surfaces were sliced off with a razor blade, demonstrating that these sponges concentrate defenses against predators in their surfaces. One semi-cryptic species and one semi-exposed species were fed upon, but not entirely consumed. By contrast, 11 of 12 of the exposed and semi-exposed species were rejected. Cryptic sponges grew out of their cavities in the reef only when protected by seasonally thick mats of macroalgae or by cages that excluded fish. Received: 10 January 1997 / Accepted: 4 February 1997     

Evidence for morphology-induced sediment settlement prevention on the tubular sponge<Emphasis Type="Italic"> Haliclona urceolus</Emphasis>

Several mechanisms are known to assist the survival of sponges in highly sedimented environments. This study considers the potential of sponge morphology and the positioning of exhalant water jets (through the osculum) in the adaptation of Haliclona urceolus to highly sedimented habitats. This sponge is cylindrical with an apical osculum, which is common in sedimented subtidal habitats at Lough Hyne Marine Nature Reserve, Cork, Ireland. Fifteen sponges were collected, preserved (killed with the structure and morphology maintained) and then replaced in a high sediment environment next to a living specimen (at 24 m). After 5 days, the sediment settled on both living and preserved sponges was collected and dried. No sediment was collected from living sponges, while preserved specimens had considerable amounts of settled sediment on their surfaces. The amount of sediment collected on these preserved specimens was significantly linearly correlated with sponge dry weight, maximum diameter and oscula width (R²>0.70, P<0.001, df=14). Observations of flow direction (using coloured dye) through H. urceolus showed that water is drawn into the sponge on its underside and exits via a large vertically pointing osculum. Sponge morphologies (shape) have often been considered as a means of passive adaptation to a number of different environmental parameters with oscula position enabling entrained flow through the sponge in high flow conditions. However, this study shows how the combination of sponge morphology (tubular shape) and positioning of the osculum may enable H. urceolus to survive in highly sedimented environments. Similar mechanisms may also aid the survival of some deep-water sponge species with similar morphologies.Communicated by J.P. Thorpe, Port Erin     

Variable effects of symbiotic snapping shrimps on their sponge hosts

Mutualistic relationships are ubiquitous in tropical coral reefs, but the costs and benefits to partner species are often poorly known. In Caribbean coral reefs, several species of snapping shrimp (Synalpheus spp.) dwell exclusively in marine sponges, which serve as both habitat and food source. A paired experimental design was used to examine the effects of Synalpheus occupancy on predation, morphology, and growth of their sponge host Lissodendoryx colombiensis in Bocas del Toro, Panama (9.351°N, 82.258°W) in June 2009. Shrimp occupancy significantly decreased consumption of sponges by a predatory sea star (Oreaster reticulatus) and also affected sponge morphology; sponges grown without shrimps decreased in canal size, in both the laboratory and the field. Shrimp occupancy had more ambiguous effects on sponge growth. In laboratory experiments, shrimp occupancy benefited sponge growth, although all sponges experienced overall decreases in mass. In field experiments, there were no significant differences in growth between occupied and empty sponges. However, the benefits of shrimp occupancy on sponge growth were negatively correlated with overall increases in sponge size; sponges that decreased in mass during the experiment benefited more from shrimp occupancy than sponges that increased in mass. These costs and benefits suggest that Synalpheus has variable effects on sponges: positive effects on sponges in the presence of predators, and/or when sponges are decreasing in mass (e.g., during periods of physical stress), but a negative effect on sponges during periods of active sponge growth.     

Microbial associations in sponges. I. Ecology,physiology and microbial populations of coral reef sponges

Illumination, current strength and physical turbulence influence the distribution of 4 tropical sponges. Three sponges with cyanobacteria in exposed tissues grow only in poen shallow habitats: Pericharax heteroraphis in moderate-current, lowturbulence regions on the reef slope; Jaspis stellifera in low-current, moderate-turbulence regions of the outer reef flat; and Neofibularia irata in moderate-current, high-turbulence areas below the reef crest. Ircinia wistarii contains no cyanobacteria and occurs in deeper, strong-current, high-turbulence regions. N. irata agressively overgrows neighbouring corals and its growth form is influenced by the current strength. The sponges efficiently filter bacteria from the water. The efficiency is related to the aquiferous structure, particularly the size of choanocyte chambers, and is unrelated to the existing bacterial populations in sponge tissue. The numbers of bacteria associated with the sponges are proportional to the sponge mesohyl density, with the dense sponges J. stellifera and I. wistarii containing many bacteria whereas P. heteroraphis is not dense and has few bacteria.     

A study of the variations in lipid levels,lipid class composition and fatty acid composition in the first stages ofArtemia sp.

The lipid class composition and the fatty acid composition of total lipids of the cysts, newly hatched nauplii and 24-h-old metanauplii of a Spanish parthenogenetic diploid strain ofArtemia sp. were studied. Substantial differences in the total lipid level occurred among these stages, with a marked increase from the cyst to the nauplii being followed by a decrease in the metanaupliar stages. This variation affected the absolute levels (mg/g dry wt) of the total lipid classes and individual fatty acids, although the percent composition of the fatty acids in total lipid was essentially unchanged. An exception occurred during hatching in that the percentages of 16:0 and 16:1n-7 in total lipid decreased whereas that of 20: 5n-3 increased. The lipid classes showed higher variation than the fatty acids both in absolute and in relative terms, and in particular, the ratio of phosphatidylcholine:phosphatidylethanolamine decreased progressively from cysts to nauplii and metanauplii. The implications of these findings for the use ofArtemia sp. as a larval feed in aquaculture are considered.     

Variation in secondary metabolite and aragonite concentrations in the tropical green seaweed Neomeris annulata: effects on herbivory by fishes

The tropical alga Neomeris annulata (Dickie) (Dasycladaceae: Chlorophyta) produces brominated sesquiterpenes and deposits aragonite throughout the thallus. This study, conducted throughout 1990–1991, showed that the fleshy, apical portions of the thalli (tips) were high in secondary metabolite concentrations (1.5%, mean combined secondary metabolites based on dry mass) and relatively low in calcium carbonate (aragonite form) (65.2% ash). The basal portions were lower in combined secondary metabolites (0.2% dry mass), and higher in aragonite (90.0% ash). The crude organic extract of the alga deterred fish feeding in the field at concentrations of 5, 10 and 15% dry mass, but not at a lower concentration of 1.5%. Natural concentrations of crude organic extract ranged from 1.5 to 15.3% in whole individuals and averaged 5.1% based on dry mass. Two brominated sesquiterpenes were isolated as major metabolites from the crude extract, but only one deterred feeding at natural concentrations. Ash concentrations in N. annulata were 60% dry mass in both the tips and bases. Aragonite strongly deterred feeding at concentrations of 65 and 90% dry mass. When a naturally occurring combination of organic extract and aragonite in the tips (10% crude extract and 65% aragonite) was compared with that of the bases (0.8% crude extract and 90% aragonite), no significant difference in grazing was observed. Combinations of secondary metabolites and aragonite were also tested against one or the other single defense. The combination of defenses proved a more effective deterrent than either secondary metabolites or aragonite alone.