A marine-derived fatty acid targets the cell membrane of Gram-positive bacteria.

IMPORTANCE: With the lack of new antibiotics in the drug discovery pipeline, coupled with accelerated evolution of antibiotic resistance, new sources of antibiotics that target pathogens of clinical importance are paramount. Here, we use bacterial cytological profiling to identify the mechanism of action of the monounsaturated fatty acid (Z)-13-methyltetra-4-decenoic acid isolated from the marine bacterium Olleya marilimosa with antibacterial effects against Gram-positive bacteria. The fatty acid antibiotic was found to rapidly destabilize the cell membrane by pore formation and membrane aggregation in Bacillus subtilis, suggesting that this fatty acid may be a promising adjuvant used in combination to enhance antibiotic sensitivity.

A bacterial quorum sensing signal is a potent inhibitor of de novo pyrimidine biosynthesis in the globally abundant Emiliania huxleyi.

Interactions between marine phytoplankton, viruses, and bacteria drive biogeochemical cycling, shape marine trophic structures, and impact global climate. Microbially produced compounds have emerged as key players in influencing eukaryotic organismal physiology, and in turn, remodel microbial community structure. This work aimed to reveal the molecular mechanism by which the bacterial quorum sensing molecule 2-heptyl-4-quinolone (HHQ), produced by the marine gammaproteobacterium Pseudoalteromonas spp., arrests cell division and confers protection from virus-induced mortality in the bloom-forming coccolithophore Emiliania huxleyi. Previous work has established alkylquinolones as inhibitors of dihydroorotate dehydrogenase (DHODH), a fundamental enzyme catalyzing the fourth step in pyrimidine biosynthesis and a potential antiviral drug target. An N-terminally truncated version of E. huxleyi DHODH was heterologously expressed in E. coli, purified, and kinetically characterized. Here, we show HHQ is a potent inhibitor (Ki of 2.3 nM) of E. huxleyi DHODH. E. huxleyi cells exposed to brequinar, the canonical human DHODH inhibitor, experienced immediate, yet reversible cellular arrest, an effect which mirrors HHQ-induced cellular stasis previously observed. However, brequinar treatment lacked other notable effects observed in HHQ-exposed E. huxleyi including significant changes in cell size, chlorophyll fluorescence, and protection from virus-induced lysis, indicating HHQ has additional as yet undiscovered physiological targets. Together, these results suggest a novel and intricate role of bacterial quorum sensing molecules in tripartite interdomain interactions in marine ecosystems, opening new avenues for exploring the role of microbial chemical signaling in algal bloom regulation and host-pathogen dynamics.

Bacterial Quorum-Sensing Signal Arrests Phytoplankton Cell Division and Impacts Virus-Induced Mortality.

Interactions between phytoplankton and heterotrophic bacteria fundamentally shape marine ecosystems by controlling primary production, structuring marine food webs, mediating carbon export, and influencing global climate. Phytoplankton-bacterium interactions are facilitated by secreted compounds; however, linking these chemical signals, their mechanisms of action, and their resultant ecological consequences remains a fundamental challenge. The bacterial quorum-sensing signal 2-heptyl-4-quinolone (HHQ) induces immediate, yet reversible, cellular stasis (no cell division or mortality) in the coccolithophore Emiliania huxleyi; however, the mechanism responsible remains unknown. Using transcriptomic and proteomic approaches in combination with diagnostic biochemical and fluorescent cell-based assays, we show that HHQ exposure leads to prolonged S-phase arrest in phytoplankton coincident with the accumulation of DNA damage and a lack of repair despite the induction of the DNA damage response (DDR). While this effect is reversible, HHQ-exposed phytoplankton were also protected from viral mortality, ascribing a new role of quorum-sensing signals in regulating multitrophic interactions. Furthermore, our data demonstrate that in situ measurements of HHQ coincide with areas of enhanced micro- and nanoplankton biomass. Our results suggest bacterial communication signals as emerging players that may be one of the contributing factors that help structure complex microbial communities throughout the ocean.IMPORTANCE Bacteria and phytoplankton form close associations in the ocean that are driven by the exchange of chemical compounds. The bacterial signal 2-heptyl-4-quinolone (HHQ) slows phytoplankton growth; however, the mechanism responsible remains unknown. Here, we show that HHQ exposure leads to the accumulation of DNA damage in phytoplankton and prevents its repair. While this effect is reversible, HHQ-exposed phytoplankton are also relieved of viral mortality, elevating the ecological consequences of this complex interaction. Further results indicate that HHQ may target phytoplankton proteins involved in nucleotide biosynthesis and DNA repair, both of which are crucial targets for viral success. Our results support microbial cues as emerging players in marine ecosystems, providing a new mechanistic framework for how bacterial communication signals mediate interspecies and interkingdom behaviors.

Characterizing the culturable surface microbiomes of diverse marine animals.

Biofilm-forming bacteria have the potential to contribute to the health, physiology, behavior and ecology of the host and serve as its first line of defense against adverse conditions in the environment. While metabarcoding and metagenomic information furthers our understanding of microbiome composition, fewer studies use cultured samples to study the diverse interactions among the host and its microbiome, as cultured representatives are often lacking. This study examines the surface microbiomes cultured from three shallow-water coral species and two whale species. These unique marine animals place strong selective pressures on their microbial symbionts and contain members under similar environmental and anthropogenic stress. We developed an intense cultivation procedure, utilizing a suite of culture conditions targeting a rich assortment of biofilm-forming microorganisms. We identified 592 microbial isolates contained within 15 bacterial orders representing 50 bacterial genera, and two fungal species. Culturable bacteria from coral and whale samples paralleled taxonomic groups identified in culture-independent surveys, including 29% of all bacterial genera identified in the Megaptera novaeangliae skin microbiome through culture-independent methods. This microbial repository provides raw material and biological input for more nuanced studies which can explore how members of the microbiome both shape their micro-niche and impact host fitness.

Identification of a bacteria-produced benzisoxazole with antibiotic activity against multi-drug resistant Acinetobacter baumannii.

The emergence of multi-drug resistant pathogenic bacteria represents a serious and growing threat to national healthcare systems. Most pressing is an immediate need for the development of novel antibacterial agents to treat Gram-negative multi-drug resistant infections, including the opportunistic, hospital-derived pathogen, Acinetobacter baumannii. Herein we report a naturally occurring 1,2-benzisoxazole with minimum inhibitory concentrations as low as 6.25 µg ml-1 against clinical strains of multi-drug resistant A. baumannii and investigate its possible mechanisms of action. This molecule represents a new chemotype for antibacterial agents against A. baumannii and is easily accessed in two steps via de novo synthesis. In vitro testing of structural analogs suggest that the natural compound may already be optimized for activity against this pathogen. Our results demonstrate that supplementation of 4-hydroxybenzoate in minimal media was able to reverse 1,2-benzisoxazole's antibacterial effects in A. baumannii. A search of metabolic pathways involving 4-hydroxybenzoate coupled with molecular modeling studies implicates two enzymes, chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyltransferase, as promising leads for the target of 3,6-dihydroxy-1,2-benzisoxazole.

Bacterial alkylquinolone signaling contributes to structuring microbial communities in the ocean.

BACKGROUND: Marine bacteria form complex relationships with eukaryotic hosts, from obligate symbioses to pathogenic interactions. These interactions can be tightly regulated by bioactive molecules, creating a complex system of chemical interactions through which these species chemically communicate thereby directly altering the host's physiology and community composition. Quorum sensing (QS) signals were first described in a marine bacterium four decades ago, and since then, we have come to discover that QS mediates processes within the marine carbon cycle, affects the health of coral reef ecosystems, and shapes microbial diversity and bacteria-eukaryotic host relationships. Yet, only recently have alkylquinolone signals been recognized for their role in cell-to-cell communication and the orchestration of virulence in biomedically relevant pathogens. The alkylquinolone, 2-heptyl-4-quinolone (HHQ), was recently found to arrest cell growth without inducing cell mortality in selected phytoplankton species at nanomolar concentrations, suggesting QS molecules like HHQ can influence algal physiology, playing pivotal roles in structuring larger ecological frameworks. RESULTS: To understand how natural communities of phytoplankton and bacteria respond to HHQ, field-based incubation experiments with ecologically relevant concentrations of HHQ were conducted over the course of a stimulated phytoplankton bloom. Bulk flow cytometry measurements indicated that, in general, exposure to HHQ caused nanoplankton and prokaryotic cell abundances to decrease. Amplicon sequencing revealed HHQ exposure altered the composition of particle-associated and free-living microbiota, favoring the relative expansion of both gamma- and alpha-proteobacteria, and a concurrent decrease in Bacteroidetes. Specifically, Pseudoalteromonas spp., known to produce HHQ, increased in relative abundance following HHQ exposure. A search of representative bacterial genomes from genera that increased in relative abundance when exposed to HHQ revealed that they all have the genetic potential to bind HHQ. CONCLUSIONS: This work demonstrates HHQ has the capacity to influence microbial community organization, suggesting alkylquinolones have functions beyond bacterial communication and are pivotal in driving microbial community structure and phytoplankton growth. Knowledge of how bacterial signals alter marine communities will serve to deepen our understanding of the impact these chemical interactions have on a global scale.

The chemical cue tetrabromopyrrole induces rapid cellular stress and mortality in phytoplankton.

Eukaryotic phytoplankton contribute to the flow of elements through marine food webs, biogeochemical cycles, and Earth's climate. Therefore, how phytoplankton die is a critical determinate of the flow and fate of nutrients. While heterotroph grazing and viral infection contribute to phytoplankton mortality, recent evidence suggests that bacteria-derived cues also control phytoplankton lysis. Here, we report exposure to nanomolar concentrations of 2,3,4,5-tetrabromopyrrole (TBP), a brominated chemical cue synthesized by marine γ-proteobacteria, resulted in mortality of seven phylogenetically-diverse phytoplankton species. A comparison of nine compounds of marine-origin containing a range of cyclic moieties and halogenation indicated that both a single pyrrole ring and increased bromination were most lethal to the coccolithophore, Emiliania huxleyi. TBP also rapidly induced the production of reactive oxygen species and the release of intracellular calcium stores, both of which can trigger the activation of cellular death pathways. Mining of the Ocean Gene Atlas indicated that TBP biosynthetic machinery is globally distributed throughout the water column in coastal areas. These findings suggest that bacterial cues play multiple functions in regulating phytoplankton communities by inducing biochemical changes associated with cellular death. Chemically-induced lysis by bacterial infochemicals is yet another variable that must be considered when modeling oceanic nutrient dynamics.

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase-thioesterase enzyme pair.

Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.

A Bacterial Quorum-Sensing Precursor Induces Mortality in the Marine Coccolithophore, Emiliania huxleyi.

Interactions between phytoplankton and bacteria play a central role in mediating biogeochemical cycling and food web structure in the ocean. However, deciphering the chemical drivers of these interspecies interactions remains challenging. Here, we report the isolation of 2-heptyl-4-quinolone (HHQ), released by Pseudoalteromonas piscicida, a marine gamma-proteobacteria previously reported to induce phytoplankton mortality through a hitherto unknown algicidal mechanism. HHQ functions as both an antibiotic and a bacterial signaling molecule in cell-cell communication in clinical infection models. Co-culture of the bloom-forming coccolithophore, Emiliania huxleyi with both live P. piscicida and cell-free filtrates caused a significant decrease in algal growth. Investigations of the P. piscicida exometabolome revealed HHQ, at nanomolar concentrations, induced mortality in three strains of E. huxleyi. Mortality of E. huxleyi in response to HHQ occurred slowly, implying static growth rather than a singular loss event (e.g., rapid cell lysis). In contrast, the marine chlorophyte, Dunaliella tertiolecta and diatom, Phaeodactylum tricornutum were unaffected by HHQ exposures. These results suggest that HHQ mediates the type of inter-domain interactions that cause shifts in phytoplankton population dynamics. These chemically mediated interactions, and other like it, ultimately influence large-scale oceanographic processes.

Enhancement of antibiotic activity against multidrug-resistant bacteria by the efflux pump inhibitor 3,4-dibromopyrrole-2,5-dione isolated from a Pseudoalteromonas sp.

Members of the resistance nodulation cell division (RND) of efflux pumps play essential roles in multidrug resistance (MDR) in Gram-negative bacteria. Here, we describe the search for new small molecules from marine microbial extracts to block efflux and thus restore antibiotic susceptibility in MDR bacterial strains. We report the isolation of 3,4-dibromopyrrole-2,5-dione (1), an inhibitor of RND transporters, from Enterobacteriaceae and Pseudomonas aeruginosa, from the marine bacterium Pseudoalteromonas piscicida. 3,4-Dibromopyrrole-2,5-dione decreased the minimum inhibitory concentrations (MICs) of two fluoroquinolones, an aminoglycoside, a macrolide, a beta-lactam, tetracycline, and chloramphenicol between 2- and 16-fold in strains overexpressing three archetype RND transporters (AcrAB-TolC, MexAB-OprM, and MexXY-OprM). 3,4-Dibromopyrrole-2,5-dione also increased the intracellular accumulation of Hoechst 33342 in wild-type but not in transporter-deficient strains and prevented H33342 efflux (IC50 = 0.79 µg/mL or 3 µM), a hallmark of efflux pump inhibitor (EPI) functionality. A metabolomic survey of 36 Pseudoalteromonas isolates mapped the presence of primarily brominated metabolites only within the P. piscicida phylogenetic clade, where a majority of antibiotic activity was also observed, suggesting a link between halogenation and enhanced secondary metabolite biosynthetic potential. In sum, 3,4-dibromopyrrole-2,5-dione is a potent EPI and deserves further attention as an adjuvant to enhance the effectiveness of existing antibiotics.

Cytochrome P450 diversity and induction by gorgonian allelochemicals in the marine gastropod Cyphoma gibbosum.

BACKGROUND: Intense consumer pressure strongly affects the structural organization and function of marine ecosystems, while also having a profound effect on the phenotype of both predator and prey. Allelochemicals produced by prey often render their tissues unpalatable or toxic to a majority of potential consumers, yet some marine consumers have evolved resistance to host chemical defenses. A key challenge facing marine ecologists seeking to explain the vast differences in consumer tolerance of dietary allelochemicals is understanding the biochemical and molecular mechanisms underlying diet choice. The ability of marine consumers to tolerate toxin-laden prey may involve the cooperative action of biotransformation enzymes, including the inducible cytochrome P450s (CYPs), which have received little attention in marine invertebrates despite the importance of allelochemicals in their evolution. RESULTS: Here, we investigated the diversity, transcriptional response, and enzymatic activity of CYPs possibly involved in allelochemical detoxification in the generalist gastropod Cyphoma gibbosum, which feeds exclusively on chemically defended gorgonians. Twelve new genes in CYP family 4 were identified from the digestive gland of C. gibbosum. Laboratory-based feeding studies demonstrated a 2.7- to 5.1-fold induction of Cyphoma CYP4BK and CYP4BL transcripts following dietary exposure to the gorgonian Plexaura homomalla, which contains high concentrations of anti-predatory prostaglandins. Phylogenetic analysis revealed that C. gibbosum CYP4BK and CYP4BL were most closely related to vertebrate CYP4A and CYP4F, which metabolize pathophysiologically important fatty acids, including prostaglandins. Experiments involving heterologous expression of selected allelochemically-responsive C. gibbosum CYP4s indicated a possible role of one or more CYP4BL forms in eicosanoid metabolism. Sequence analysis further demonstrated that Cyphoma CYP4BK/4BL and vertebrate CYP4A/4F forms share identical amino acid residues at key positions within fatty acid substrate recognition sites. CONCLUSIONS: These results demonstrate differential regulation of CYP transcripts in a marine consumer feeding on an allelochemical-rich diet, and significantly advance our understanding of both the adaptive molecular mechanisms that marine consumers use to cope with environmental chemical pressures and the evolutionary history of allelochemical-metabolizing enzymes in the CYP superfamily.

The role of multixenobiotic transporters in predatory marine molluscs as counter-defense mechanisms against dietary allelochemicals.

Multixenobiotic transporters have been extensively studied for their ability to modulate the disposition and toxicity of pharmacological agents, yet their influence in regulating the levels of dietary toxins within marine consumers has only recently been explored. This study presents functional and molecular evidence for multixenobiotic transporter-mediated efflux activity and expression in the generalist gastropod Cyphoma gibbosum, and the specialist nudibranch Tritonia hamnerorum, obligate predators of chemically defended gorgonian corals. Immunochemical analysis revealed that proteins with homology to permeability glycoprotein (P-gp) were highly expressed in T. hamnerorum whole animal homogenates and localized to the apical tips of the gut epithelium, a location consistent with a role in protection against ingested prey toxins. In vivo dye assays with specific inhibitors of efflux transporters demonstrated the activity of P-gp and multidrug resistance-associated protein (MRP) families of ABC transporters in T. hamnerorum. In addition, we identified eight partial cDNA sequences encoding two ABCB and two ABCC proteins from each molluscan species. Digestive gland transcripts of C. gibbosum MRP-1, which have homology to vertebrate glutathione-conjugate transporters, were constitutively expressed regardless of gorgonian diet. This constitutive expression may reflect the ubiquitous presence of high affinity substrates for C. gibbosum glutathione transferases in gorgonian tissues likely necessitating export by MRPs. Our results suggest that differences in multixenobiotic transporter expression patterns and activity in molluscan predators may stem from the divergent foraging strategies of each consumer.

Biochemical warfare on the reef: the role of glutathione transferases in consumer tolerance of dietary prostaglandins.

BACKGROUND: Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla. PRINCIPAL FINDINGS: Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A(2) (PGA(2)) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA(2)) were also the most potent inhibitors. In vivo estimates of PGA(2) concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA(2) and operating at or near physiological capacity. SIGNIFICANCE: The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may operate as 'all-purpose' detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.

The emerging role of pharmacology in understanding consumer-prey interactions in marine and freshwater systems.

Within our lakes, streams, estuaries, and oceans, there is an astounding chemodiversity of secondary metabolites produced by microbes, algae, and invertebrates. Nearly 30 years of study have yielded hundreds of examples in which secondary metabolites alter the foraging behavior or fitness of aquatic consumers, or both. However, our understanding of the mechanisms that mediate the fate and consequences of these metabolites in aquatic consumers remains in its infancy. Interactions between metabolites and consumers at the molecular and biochemical level are the purview of modern pharmacology, which is rooted in the long history of human-drug interactions and can be adopted for ecological studies. Here, we argue that a pharmacological approach to consumer-prey interactions will be as productive within aquatic systems as it has been for understanding terrestrial systems. We review the diversity of secondary metabolites in aquatic organisms, their known effects on the feeding behaviors and performance of aquatic consumers, and the few studies that have attempted to describe their biochemical manipulation within consumer tissues, i.e., their absorption, distribution, metabolism (including detoxification), and excretion. We then highlight vexing issues in the ecology and evolution of aquatic consumer-prey interactions that would benefit from a pharmacological approach, including specialist-versus-generalist feeding strategies, dietary mixing, nutrient-toxin interactions, and taste. Finally, we argue that a pharmacological approach could help to predict how consumer-prey interactions are altered by global changes in pH, water temperature and ultraviolet radiation, or by pollution. Arguably, the state of knowledge of aquatic consumer-prey interactions is equivalent to that faced by ecologists studying terrestrial herbivores in the 1970s; the literature documents profound variation among consumers in their feeding tolerances for secondary metabolites without a thorough understanding of the mechanisms that underlie that variation. The subsequent advancement in our understanding of terrestrial herbivores in the intervening decades provides confidence that applying a pharmacological approach to aquatic consumers will prove equally productive.

Proteomic identification, cDNA cloning and enzymatic activity of glutathione S-transferases from the generalist marine gastropod, Cyphoma gibbosum.

Glutathione S-transferases (GST) were characterized from the digestive gland of Cyphoma gibbosum (Mollusca; Gastropoda), to investigate the possible role of these detoxification enzymes in conferring resistance to allelochemicals present in its gorgonian coral diet. We identified the collection of expressed cytosolic Cyphoma GST classes using a proteomic approach involving affinity chromatography, HPLC and nano-spray liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two major GST subunits were identified as putative mu-class GSTs; while one minor GST subunit was identified as a putative theta-class GST, apparently the first theta-class GST identified from a mollusc. Two Cyphoma GST cDNAs (CgGSTM1 and CgGSTM2) were isolated by RT-PCR using primers derived from peptide sequences. Phylogenetic analyses established both cDNAs as mu-class GSTs and revealed a mollusc-specific subclass of the GST-mu clade. These results provide new insights into metazoan GST diversity and the biochemical mechanisms used by marine organisms to cope with their chemically defended prey.

Multiple defensive roles for triterpene glycosides from two Caribbean sponges.

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.