Despite their high nutritional value and a lack of physical defenses, most marine sponges appear to be minimally affected by predators, competitors, and fouling organisms, possibly due to sponge chemical defenses. In the last 15 years, several triterpene glycosides have been isolated from sponges, but their ecological or physiological roles are largely unknown. We tested triterpene glycosides from Erylus formosus and Ectyoplasia ferox, Caribbean sponges belonging to two different orders, in field and laboratory assays for effects on fish feeding, attachment by potential biofilm-forming bacteria, fouling by invertebrates and algae, and overgrowth by neighboring sponges. Formoside and other triterpene glycosides from Erylus formosus deterred predation, microbial attachment, and fouling by invertebrates and algae. Triterpene glycosides from Ectyoplasia ferox were found to be antipredatory and allelopathic. Thus, triterpene glycosides in these sponges appear to have multiple ecological functions. Tests with different triterpene glycosides at several concentrations indicated that small differences in molecular structure affect ecological activity. In order to establish whether triterpene glycosides could be involved in water-borne versus surface-mediated interactions, the presence of triterpene glycosides in the seawater surrounding live sponges was measured using two in situ sampling methods followed by HPLC and NMR spectral analysis. Water-borne triterpene glycosides were below detection limits for both species. However, top sponge layers and swabs of the surfaces of both sponges contained sufficiently high concentrations of triterpene glycosides to deter bacterial settlement and fouling of Erylus formosus surfaces and overgrowth of Ectyoplasia ferox by neighboring sponges. Enemies of these sponges appear to be deterred by surface contact of triterpene glycosides rather than by water-borne interactions. The dual strategy of employing one group of compounds for multiple purposes and minimizing the loss of compounds into seawater suggests that these organisms utilize chemical defenses with efficiency.

Attachment is one of the first steps in bacterial colonization. By inhibiting bacterial attachment on surface cells, sponges may not only prevent infection, but also the process of biofouling. Crude organic extracts from 26 species of Caribbean sponges were assayed for their ability to inhibit bacterial attachment. Bacterial attachment was tested using Vibrio harveyi, a motile marine bacterium, isolated from seawater collected above one of the reefs from which sponges were sampled. Extracts were incorporated into agar blocks at concentrations volumetrically equivalent to whole sponge tissue. Extracts from 21 of 26 species (81%) resulted in bacterial attachment on treated blocks that was < 40% of attachment on controls. Of these extracts, 9 were particularly active, with mean levels of attachment < 8% of controls (Agelas conifera, Ailochroia crassa, Aka coralliphagum, Amphimedon compressa, Aplysina fulva, Erylus formosus, Plakortis halichondroides, Ptilocaulis spiculifera, Verongula gigantea). Extracts from 4 species (Ailochroia crassa, Chondrilla nucula, Ectyoplasia ferox, and Iotrochota birotulata) inhibited bacterial attachment in this assay but were not found to inhibit bacterial growth in a previous study. Purified compounds that deterred feeding of predatory fishes in a prior study were also tested for their effects on bacterial attachment; they were: oroidin, 4, 5-dibromopyrrole-2-carboxylic acid and sceptrin from Agelas species, amphitoxin from A. compressa, aeroplysinin-1 and dibromocyclohexadienone from Aplysina species, steroidal glycosides from E. ferox, and formoside from E. formosus. Of these, all but the steroidal glycosides from E. ferox deterred bacterial attachment at natural concentrations, providing evidence that sponge secondary metabolites may have multiple ecological functions.

Crude organic tissue extracts from 8 species of Caribbean sponges were assayed for inhibitory effects on surface colonization using 24 environmental marine bacterial isolates, 4 known marine invertebrate pathogens, and 1 common fouling bacterium. Each extract was tested for its effects on bacterial attachment, growth and swarming. The 24 bacterial strains were isolated from sponge surfaces, nearby substrata, or adjacent seawater. Extracts were incorporated into agar for assays of bacterial attachment and swarming. Growth-inhibition assays were conducted with the standard agar disk-diffusion assay. Of the 24 bacterial isolates, 23 were significantly inhibited from attaching to an extract-treated agar surface; 1 isolate from the surface of Agelas conifera exhibited significantly enhanced attachment on agar treated with the extract of that sponge. Sponge extracts had the least effect on growth: of 184 assays, 11 displayed significant antibacterial activity, all of these from 4 sponge species (A. conifera, Ailochroia crassa, Amphimedon compressa, and Aplysina fulva). The same isolate from the surface of A. conifera that exhibited enhanced attachment in response to the extract of that sponge exhibited inhibited growth in response to the same extract. Six out of 24 bacterial isolates exhibited swarming, the majority (67%) of which were isolated from substratum sources. Extracts from 4 of the 8 sponge species (the same species as listed above) inhibited swarming in all 6 strains, while the remaining extracts enhanced, inhibited, or had no effect on swarming depending on the strain. Bioassay-guided fractionation of the extract of A. crassa yielded 2 compounds responsible for inhibiting attachment and swarming, respectively. Ianthellin was identified as the metabolite that inhibited attachment, whereas another brominated tryosine metabolite inhibited swarming. Chemical defenses of sponges may target microbial attachment, and to a lesser degree influence swarming and growth. Non-toxic metabolites may play the greatest role in affecting bacterial epibiosis on the surfaces of marine sponges.