Journal of Chemical Ecology, VoL 18, No. 3, 1992

CHEMISTRY A N D CHEMICAL ECOLOGY OF THE BAHAMIAN SPONGE Aplysilla glacialis 1

STEVEN

C. BOBZIN*

and D. JOHN

FAULKNER

Scripps Institution of Oceanography University of California, San Diego Mail Code 0212F, La Jolla, California 92093-0212 (Received July 29, 1991; accepted October 28, 1991)

Abstract--Chemical investigation of the secondary metabolites of the marine sponge Aplysilla glacialis collected at French Wells, Crooked Island, Bahamas, resulted in the isolation and characterization of four diterpenes, two sterol endoperoxides, and two methylated primary metabolites. Some of these compounds, along with crude extracts of the sponge, were investigated for their ability to deter fish predation, affect the fouling of surfaces, and inhibit the growth of marine microorganisms. The diterpene mano/51 (3), cholesterol endoperoxide (4), and the crude nonpolar extract of A. glacialis were shown to deter feeding by a natural assemblage of fish predators in an in situ assay conducted at French Wells. Pure secondary metabolites and crude extracts of A. glacialis also were tested in a laboratory fish feeding assay employing the wrasse Thalassoma lunare. A mixture of sterol endoperoxides was isolated from the mucus that coats the surface of A. glacialis and is exuded in large quantities when the sponge is disturbed. These compounds are thereby distributed in a manner in which they can best serve a defensive role for the sponge. An in situ assay was designed to determine the effect that pure secondary metabolites and crude extracts have on the fouling of surfaces. Manor1 (3) and cholesterol endoperoxide (4) were determined to increase the rate of fouling when compared to control surfaces. 1-Methyladenine (5) was identified as an antimicrobial constituent of A. glacialis that inhibited the growth of four marine bacteria isolated from seawater samples collected at French Wells. * To whom correspondence should be addressed at: University of Hawaii, Department of Chemistry, 2545 The Mall, Honolulu, Hawaii 96822. 1This work was presented at the Gordon Research Conferences (Marine Natural Products Chemistry), February 1990, Oxnard, California. 309 0098-0331/9210300-0309506.50/0 9 1992 Plenum Publishing Corporation

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Key Words--Aplysilla glacialis, Dendroceratida, marine sponge, cholesterol endoperoxide, sterol endoperoxides, mano61, 1-methyladenine, marine chemical ecology, fish feeding deterrence, fouling deterrence, antifouling.

INTRODUCTION

Marine sponges have proven to be an extremely rich source of natural products (see reviews by Minale et al., 1976; Faulkner, 1984, 1986, 1987, 1988, 1990, 1991). Sponge secondary metabolites provide examples of many unique carbon skeletons and include compounds derived from terpenoid, amino acid, alkaloid, acetogenin, and mixed biosynthetic origins. Most of the interest in natural products from sponges has come from the desire to isolate new compounds with potential pharmaceutical applications. Sponge metabolites have been shown to possess hypotensive (Baslow, 1977), antiinflammatory (Kernan, 1988; Glaser et al,, 1989), antiviral (Rinehart et al., 1981), cytotoxic (Rinehart et al., 1981; Jacobs et at., 1981), antimicrobial (Rinehart et al., 1981), antifungal (Baker and Wells~ 1981), and many other potentially useful pharmacological properties (Baker and Wells, 1981; Kaul, 1982). To a much lesser extent, studies have been undertaken to determine the roles that these chemicals may play in the natural environment. Many of the reports of secondary metabolites from sponges include speculation regarding the defensive purposes of the compounds isolated, but there has been relatively little effort to experimentally determine the natural functions of sponge metabolites. There are many ecological roles that may be played by secondary metabolites in the marine environment. Conspecific and interspecific interactions are often mediated by the presence of secondary metabolites in ways that enhance the survival of the producing organism. Most of these aspects have been discussed in recent reviews on marine chemical ecology (Bakus et al., 1986; Van Alstyne and Paul, 1989; Coll and Sammarco, 1989). Secondary metabolites may provide a competitive advantage for a sponge by deterring predation by fish or invertebrates, deterring settlement or overgrowth by other benthic organisms competing for substrate space, preventing invasion by pathogenic microorganisms, and attracting conspecific gametes. Our continuing interest in the chemistry of sponges of the order Dendroceratida (Bobzin and Faulkner, 1989a, b, 1991a, b; Bobzin, 1990) led us to collect the pink encrusting sponge Aplysilla glacialis from the mangrove lagoon at French Wells, Crooked Island, Bahamas, in June 1988 and July 1989, Aplysilla glacialis is a cosmopolitan species that has been reported from the Caribbean, North and South America, Australia, and the Arctic Ocean (Zea, 1987). Our investigation of sponges of the order Dendroceratida was motivated by the hypothesis that the absence of siliceous spicules, which is characteristic of this

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order, may be compensated by an increase in the variety and quantity of defensive secondary metabolites produced by these animals. This hypothesis originates from the observation that the loss of a physical defense mechanism, such as spicules, may be the result of an evolutionary development of an alternate, chemical defense mechanism (Faulkner and Ghiselin, 1983). Aplysilla glacialis was found only on the prop roots of the mangrove trees that line the lagoon at French Wells in depths of 0-2 m. Aplysilla glacialis is rarely overgrown by the invertebrates and algae that compete for substrate space in this diverse environment, and it was never observed to be eaten by fish despite the fleshy, aspiculous nature of its tissue. In situ and laboratory assays were designed to determine whether crude extracts of A. glacialis, or pure secondary metabolites isolated from these extracts, were responsible for these observations. The assays employed in this work were designed to determine whether the extracts or metabolites deterred feeding by fish, affected the rate of fouling of surfaces, and inhibited the growth of marine microorganisms. A sample of Aplysilla glacialis collected in British Columbia has been reported to possess a number of spongian diterpene metabolites (Tischler and Anderson, 1989; Tischler et al., 1991). Two of these diterpenes, glaciolide (1) and cadlinolide A (2), were also isolated from the dorid nudibranch Cadlina luteomarginata, which was collected in the same locality. It was suggested that the glaciolide and cadlinolide A found in C. luteomarginata were obtained by the nudibranch from its diet, which includes A. glacialis. Dorid nudibranchs often sequester secondary metabolites from their sponge diets, and these compounds are widely believed to serve a defensive role for both animals. Unfortunately, no effort was made to determine whether glaciolide (1) and cadlinolide A (2) served any ecological role for the sponge and the nudibranch. Chemical investigation of our samples of Aplysilla glacialis collected at French Wells, Crooked Island, Bahamas, did not result in the isolation of any of the spongian diterpenes previously reported from this species. Instead, four diterpenes, two sterol endoperoxides, and two methylated primary metabolites were isolated from the Bahamian collections made during 1988 and 1989. Crude extracts and the pure metabolites mano61 (3) and 5a,8~-epidioxycholest-6-en313-ol (4) ("cholesterol endoperoxide") obtained from the June 1988 collection of A. glacialis were assayed in San Diego Bay, California, in January 1990 to determine their effect on the fouling of surfaces in situ. Mano61 (3), cholesterol endoperoxide (4), and the dichloromethane-soluble portion of the fresh methanol extract of A. glacialis were tested for their effect on fish feeding in an in situ assay carried out at French Wells in July 1989. Crude extracts and pure secondary metabolites isolated from a sample of A. glacialis collected in July 1989 were tested in a laboratory fish feeding deterrence assay employing the wrasse Thalassoma lunare. These crude extracts and pure compounds were also screened for antimicrobial activity against nine bacterial isolates obtained from seawater

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samples collected in the mangrove lagoon at French Wells in July 1989. 1-Methyladenine (5) was isolated as an antimicrobial metabolite from A. g/acialis that inhibited the growth of four of the nine marine bacterial isolates screened. Several other secondary metabolites were also isolated from the collections of A. glacialis, but these compounds were not obtained in large enough quantity to allow testing in these assays. METHODS AND MATERIALS

Collection of Aplysilla glacialis. The pink encrusting sponge A. glacialis was collected by hand at a depth of 0-2 m from the mangrove prop roots in the lagoon at French Wells, Crooked Island, Bahamas (Lat. 22~ Long. 74~ in June 1988 (sample 88-275) and July 1989 (samples 89-150B and 89-150C). Numerous individuals were combined in each of these collections. The samples were either stored frozen ( - 1 0 ~ or directly extracted in methanol. Extraction and Chromatography ofAplysilla glacialis. The sponge (sample 88-275) was stored frozen for approximately six months and then freeze-dried. The lyophilized sponge tissue (54.4 g) was successively extracted with hexane (! • 600 ml), hexane-dichloromethane (1 : 1, 2 • 800 ml), dichloromethane (4 x 600 ml), ethyl acetate (1 x 600 ml), and methanol (4 x 600 ml). The hexane and dichloromethane extracts were combined to yield a crude nonpolar extract (1.9 g, 3.5% dry weight) that was separated by flash chromatography on silica2 (column size 39 x 4 cm) using a solvent gradient from hexane-ethyl acetate (9: 1) to ethyl acetate to elute the mixture. A fraction (42 mg) that eluted with hexane-ethyl acetate (4: 1) was purified by HPLC on #-Partisil in hexanediethyl ether (4:1) to yield mano61 (3, 21.0 mg, 0.039% dry weight). Mano61 (3) was identified by comparison of the [13C]NMR data with published values (Buckwalter et al., 1975). A second fraction (157 mg) that eluted with hexaneethyl acetate (2 : 3) was separated by HPLC on/~-Partisil in hexane-ethyl acetate (1:1) to yield two nearly pure sterol endoperoxides3 along with a mixture of several other sterol endoperoxides. Both of the nearly pure fractions (34 and 35 rag) were purified by HPLC on/z-Partisil in hexane-ethyl acetate (1 : 1) to yield 5a,8ot-epidioxy-24~-ethylcholest-6-en-3/3-ol (6, 33.8 mg, 0.062% dry weight) ("24-ethylcholesterol endoperoxide") and 5a,8ot-epidioxycholest-6-en-3/3-ol (4, 33.2 mg, 0.061% dry weight) (cholesterol endoperoxide), respectively. These two compounds were identified by their characteristic [~H]NMR resonances and 2Kieselgel 60, 230-400 mesh (Gallade Chemical, Escondido, California)was used for all silica flash chromatographyseparations(Still et al., 1978). 3We definethe term "sterol endoperoxide"as any compoundpossessinga tetracyclicsterolskeleton (cf. cholesterol)and an endoperoxidebridgebetweencarbons 5 and 8.

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fragments in the low-resolution mass spectrum (Gunatilaka et al., 1981). The ethyl acetate and methanol extracts were combined to form a polar extract (10.3 g, 19.0% dry weight). This polar extract was triturated with dichloromethane (3 • 100 ml) to yield a polar triturate (1.2 g, 2.2% dry weight). The second collection of Aplysilla glacialis (sample 89-150B, 6.5 g dry weight) was directly extracted in methanol (3 • 250 ml). The methanol extract was partitioned between dichloromethane (2 • 200 ml) and water (200 ml), the organic soluble portion dried over sodium sulfate, and the solvent removed to yield a green oil (202 mg, 4.1% dry weight) that exhibited antimicrobial activity against Bacillus subtilis and Staphylococcus aureus. This mixture was separated on Sephadex LH-20 (column size 95 • 3 cm) in dichloromethane-methanol (1 : 1). The active fractions were combined and separated by flash chromatography on silica (column size 17.5 • 1.7 cm) using a solvent gradient from hexane-ethyl acetate (9: 1) to ethyl acetate to elute the mixture. This procedure yielded an impure sample of mano61 (3, 12.7 mg, 0.26% dry weight), a mixture of sterol endoperoxides (11.7 mg, 0.24% dry weight), and two fractions that appeared by [1H]NMR to consist of diterpenes that had not been isolated from the previous sample (88-275) of A. glacialis. The least polar of these two fractions (12 mg) was separated by HPLC on/~-Partisil in hexane-diethyl ether (7 : 3) to yield spongia-16-one (7, 1.9 mg, 0.039% dry weight). Spongia-16-one (7) was identified by comparison on the [1H]- and [13C]NMR data with published values (Keman et al., 1990). The second fraction (16 mg) was purified by HPLC on #-Partisil in hexane-ethyl acetate (1:1) to yield atisane-3/3,16c~-diol (8, 4.1 mg, 0.083% dry weight). Atisane-3/3,16c~-diol (8) crystallized from diethyl ether-methanol and its structure was determined by single-crystal X-ray diffraction analysis. The third collection of Aplysilla glacialis (sample 89-150C) was stored frozen for approximately three months and then freeze-dried. The lyophilized sponge tissue (8.8 g) was extracted successively with hexane (3 • 100 ml), dichloromethane (3 • 100 ml), ethyl acetate (3 • 100 ml), and methanol (3 x 100 ml). The hexane extract (176 mg, 2.0% dry weight) was separated by flash chromatography on silica (column size 17 • 2 cm) using a solvent gradient from hexane to hexane-ethyl acetate (3 : 7) to elute the mixture. This produced an impure sample of mano61 (3, 8.6 mg, 0.098% dry weight), a mixture of sterol endoperoxides (8.2 mg, 0.093 % dry weight), and pure spongia-15c~, 16adiacetate (9, 43.5 mg, 0.49% dry weight). The diacetate 9 was identified by comparison of the [1H]- and [13C]NMR data with published values (Cimino et al., 1982). The dichloromethane extract (141 mg, 1.6% dry weight) was determined by [IH]NMR to contain a mixture of sterol endoperoxides and fatty acids. The ethyl acetate extract (17.1 mg, 0.19% dry weight) appeared by [IH]NMR to contain mostly fatty acids. Separation of the methanol extract of sample 89-150C was bioassay-directed

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by using the antimicrobial activity against Flavobacterium sp. (strain H-73) in the standard disk assay (Acar, 1980). The methanol extract (1.66 g, 18.8% dry weight) was separated on Sephadex LH-20 (column size 95 • 3 cm) in methanol, the bioactive fractions combined, and the solvent removed. The resulting white solid (76 rag) was triturated with dry ethyl acetate (7 ml) and dry methanol (7 rrfl) to yield N,N'-dimethylglycine bisulfite (10, 9.7 mg, 0.11% dry weight) as an inactive, insoluble component of the mixture. N,N'-Dimethylglycine bisulrite (10) was identified from [1H]NMR, [13C]NMR, and mass spectral data. The soluble, active portion was partitioned between hexane (25 ml) and methanol (25 ml). The methanol-soluble portion retained the antimicrobial activity and was further partitioned between ethyl acetate (25 ml) and water (25 ml). The water-soluble portion was triturated with dry methanol (10 ml) to yield methanolsoluble and -insoluble fractions that both retained strong activity against Flavobacterium sp. The methanol-insoluble fraction was identified as pure 1-methyladenine (5, 3.6 mg, 0.04% dry weight), while the methanol-soluble fraction was identified as impure 1-methyladenine (45.5 mg, 0.52 % dry weight). 1-Methyladenine (5) was identified from [~H]NMR, [13C]NMR, UV, and mass spectral data (Brookes and Lawley, 1960). Extraction and Chromatography of Aplysilla glacialis Mucus. A solution of seawater and mucus (ca. 250 ml) that was recovered from the plastic bags in which A. glacialis (sample 89-150C) had been collected was separated from the sponge tissue and stored frozen (-10~ for one week. The solution was freeze-dried, and the lyophilized residue was extracted with dry dichloromethane-methanol (1:1, 3 • 100 ml). This extract was partitioned between dichloromethane (75 ml) and water (75 ml), the organic soluble portion dried over sodium sulfate, and the solvent removed to yield a yellow oil (6.7 mg). This oil was separated on a silica Sep-pak (Waters Associates, Milford, Massachusetts) using hexane-ethyl acetate (1: 1) to elute the mixture. The fractions that appeared by TLC to possess sterol endoperoxides were combined and separated by HPLC on/z-Partisil in hexane-ethyl acetate (3:2) to yield a clean mixture of sterol endoperoxides (0.69 mg, 10.3% of CH2C12 solubles). TLC and [1H]NMR analyses were used to identify the mixture, but the GC-MS data were too complex to identify specific sterol endoperoxides from this mixture. Detection of Secondary Metabolites Exuded by Aplysilla glacialis. A sample of A. glacialis was collected intact by cutting off a 6-in. length of mangrove prop root upon which a relatively large specimen of the sponge was growing. The sponge-encrusted root was placed in a 32-oz jar underwater to avoid air exposure and left undisturbed for approximately 24 hr. After this length of time, the root and sponge were removed and the seawater (ca. 650 ml) filtered through three (ca. 220 ml each) disposable C18 mini-extraction columns (6 ml HC Bakerbond spe*, J.T. Baker Inc., Phillipsburg, New Jersey). Each column then was washed successively with methanol (25 ml) and dichloromethane (25 ml), the

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315

respective washes combined, the solvents removed, and the residues stored at 0~ After 10 days the methanol wash was triturated with dry methanol (4 ml) and filtered through a cotton plug to yield a white solid (46.2 mg). The dichloromethane wash was dissolved in dry dichloromethane (4 ml) and filtered through a cotton plug to yield a yellow oil (5.6 mg). TLC and [IH]NMR analysis of the two washes indicated that they contained mostly fatty acids and salt. None of the secondary metabolites repotted from samples ofA. glacialis were detected. This procedure was repeated and again failed to detect any of the secondary metabolites from A. glacialis. Fish Feeding Deterrence Assays. In situ fish feeding deterrence assays were carried out in July 1989 in the mangrove lagoon at French Wells, Crooked Island, Bahamas, in the same locations where Aplysilla glacialis was found. The assays were carried out in a manner similar to that previously described (Harvell et al., 1988; Pawlik and Fenical, 1989). Artificial fish food strips (5 • 1 • 0.5 cm) were prepared from a mixture of carrageenan agar (Gelcarin FF 961L, FMC Corp., Philadelphia, Pennsylvania), water, and a homogenate of tuna to make the food palatable to fish. The tuna homogenate was prepared from canned chunk light tuna packed in oil (6.5 oz) and water (250 ml). Batches of 10 food strips were prepared by stirring the carrageenan agar (4.0 ml) and tuna homogenate (10 ml) into water (40 ml) and heating the mixture to boiling. Dichloromethane solutions (1.5 ml) of manorl (3), cholesterol endoperoxide (4), or the dichloromethane-soluble portion of the crude methanol extract of A. glacialis were then stirred into this mixture to prepare experimental samples with a concentration of 1.6% dry weight (0.37 % wet weight) of the pure metabolites or a "natural concentration" (see below) of the dichloromethane extract. Control strips were prepared by adding an equal volume of dichloromethane to the food preparation. The hot, fluid agar mixture was then poured into a plastic mold (10 • 5 • 0.5 cm) crossed by five lengths (40 cm) of cotton string. After the mixture cooled and solidified, ten strips were sliced to size (5 • 1 • 0.5 cm), leaving lengths of string (15 cm) free from one end to be used to secure the strips to a substrate. The manorl (3) that was used in the in situ feeding assay was obtained from a commercial source (Aldrich Chemical Co., Inc., Milwaukee, Wisconsin), while cholesterol endoperoxide (4) was prepared by singlet oxygen addition to 7-dehydrocholesterol obtained from Aldrich. 7-Dehydrocholesterol (5.1 g) was dissolved in absolute ethanol (600 m!) and oxygen gas bubbled through the solution. A solution of 10% eosin in absolute ethanol (20 ml) was added as a photosensitizer, and the reaction mixture was irradiated with a 500-W tungsten lamp. The reaction mixture was stirred and refluxed for 5.0 hr, whereupon more 10% eosin in ethanol solution (20 ml) was added and the reaction was continued for an additional 7.5 hr. The solvent was removed and the reaction mixture was separated by flash chromatography on ,silica (column size 34 • 4.5 cm) using

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a solvent gradient from hexane-ethyl acetate (4: 1) to ethyl acetate to elute the material. Cholesterol endoperoxide (4, 1.77 g, 35% yield) was crystallized (MeOH, mp 149-152~ from the fractions that eluted in hexane-ethyl acetate (3:7 and 1 : 4). The dichloromethane-soluble portion of the crude methanol extract was prepared by extracting a volume of Aplysilla glacialis tissue that was equal to the volume of the fish food preparation used in the assay, therefore providing the experimental samples with a volumetric concentration of extract equal to that found in the sponge. A portion (ca. 80 ml) of A. glacialis tissue (sample 89-150B) was extracted with methanol (150 ml) for 24 hr. The methanol extract was partitioned between dichloromethane (125 ml) and water (125 ml), the organic-soluble portion dried over sodium sulfate, and the solvent removed to obtain a yellow oil. Half of this material was dissolved in dichloromethane (1.5 ml) and added to the fish food preparation as described above. Control and experimental food strips were deployed in pairs using the string to tie them directly to the mangrove prop roots (0 to - 2 m) at various locations in the lagoon at French Wells where AplysiUa glacialis was found. The strips were retrieved after 2-8 hr depending on the predator pressure at each time and place of deployment. Each strip was then measured to determine the amount consumed by estimating the percentage of the area of each 5 x 1 • 0.5 cm strip that had been eaten. The two-tailed Wilcoxon paired-sample test (Zar, 1984) was used to analyze the assay results after first excluding pairs where both the experimental and control samples had been either completely consumed or not eaten at all. Laboratory fish feeding assays were carried out in a similar manner to that described for the determination of the feeding deterrent properties of metabolites isolated from the intertidal limpet Collisella limatula (Pawlik et al., 1986). The assay animals, a group of nine wrasses (Thalassoma lunare), were kept in separate cubicles in flowing seawater aquaria. Thalassoma lunare, an IndoPacific species, was used due to the unavailability of an endemic Caribbean species. Freeze-dried krill (4-10 mg, Euphausia pacifica, Aqua-Stock Inc., Bayonne, New Jersey) were weighed and then injected with solutions of crude extracts or pure secondary metabolites from Aplysilla glacialis to produce experimental food samples of the appropriate concentration. Control food samples were prepared by only injecting the corresponding solvent into the 1o511. The solvents were then evaporated using a stream of nitrogen. Experimental and control food samples were fed in pairs to each fish individually, randomly alternating the order of control and experimental samples. A sample was determined to be rejected if the fish repeatedly (three times) took the food particle into its mouth and regurgitated it. Extracts ofAplysilla glacialis (sample 89-150C) were used in the laboratory fish feeding assay. The hexane, dichloromethane, and ethyl acetate extracts of

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A. glacialis were assayed at a concentration of 100/~g extract/mg krill, while the methanol extract was assayed at a concentration of 200/zg extract/mg krill. The pure secondary metabolites mano61 (3), cholesterol endoperoxide (4), and spongia-15ot, 16o~-diacetate (9) were assayed at a concentration of 50 ttg metabolite/mg krill. The results from these experiments were analyzed using the G test for independence (Sokal and Rohlf, 1969). In Situ Fouling Assay. The fouling assay design employed unglazed ceramic plates (2 x 2 in. surface area 55.6 cm 2) that were coated with crude extracts or pure metabolites from AplysiUa glacialis (sample 88-275) by immersing the plates in a solution of a known concentration of the extract or compound for approximately 1 min. The semiporous plates were found to absorb approximately 0.3 ml of solvent in this length of time, thereby providing an estimate of the surface concentration achieved by this method. Sample plates were backextracted to verify the exact surface concentration of each extract or pure metabolite assayed. Control plates were prepared by immersing the plate in solvent only. The plates were then suspended in the water column by hanging them from wooden dowels (36 in. long x 1/2 in. diam.) with Dacron fishing line (50 lb test, 24 in.) tied through holes (1/16 in.) that had been drilled in the plates. The dowels were suspended at a depth of about 3 ft, leaving the plates at a depth of approximately 5 ft. Each experiment consisted of a group of five experimental plates and one control plate hung in random order 6 in. apart along the wooden dowel. Plates were immersed in solutions of mano61 (3, 50 mg/ml dichloromethane), cholesterol endoperoxide (4, 40 mg/ml dichloromethane), the nonpolar extract (22 mg/ml dichloromethane), polar extract (133 mg/ml methanol), and the polar triturate (55 mg/ml dichloromethane) of Aplysilla glacialis (sample 88-275) to produce surface concentrations of 241,203, 199, 585, and 300/~g/ cm 2, respectively. Surface concentrations were checked by back-extracting (3 • 20 ml) sample plates with their respective solvent to determine the amount of material that had been adsorbed to the plate. Sample plates were also backextracted (3 x 20 ml) with their respective solvent after being left in beakers of sterile, filtered seawater (120 ml) for approximately 24 hr in order to determine the extent to which the compounds were desorbed off of the plate's surface when immersed in water. Mano61 (3, 89.5% recovery) and cholesterol endoperoxide (4, 94.1% recovery) were only slightly affected by immersion in seawater, while the nonpolar extract (13 % recovery), polar extract (3 % recovery), and polar triturate (21% recovery) were substantially desorbed off of the plate's surface over this period of time. The mano61 (3) used in this assay was obtained from a commercial source (Aldrich Chemical Co., Inc., Milwaukee, Wisconsin), while cholesterol endoperoxide (4) was prepared by singlet oxygen addition to 7-dehydrocholesterol (see above). Nineteen experiments were deployed under a boat dock at Coronado Cays

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in southwest San Diego Bay in January 1990. Ten experiments were retrieved after a period of two weeks, while the remaining nine experiments were retrieved after five weeks. The plates were preserved by rinsing them in sterile, filtered seawater and then stored individually in plastic Petri dishes (9 cm diam.) at - 1 0 ~ until analyzed. The plates were analyzed for total adsorbed protein using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, Illinois) (Smith et al., 1985) to provide an indication of the total biomass that was present on each plate. Each plate was ultrasonically agitated (0.5 hr) in a solution of 1.0 M sodium hydroxide (10 ml) to desorb the particulate matter from the plate surface. The protein from each plate was then extracted by heating the plate in this solution for half an hour at 80~ in a water bath. This technique was similar to that used to extract protein from plankton and seawater samples for quantitative analysis (Mayzaud and Martin, 1975; Barlow and Swart, 1981). After adjusting the volume back to 10 ml with distilled water, the solutions were centrifuged (15 min), and an aliquot (1.00 ml) of each of the sodium hydroxide extracts was neutralized with 1.0 M hydrochloric acid (1.00 ml). An aliquot (0.20 ml) of each neutralized assay solution was mixed with the BCA working reagent (4.00 ml) and the solutions incubated for 30 min in a water bath at 60~ Each sample was prepared in duplicate. After the samples were cooled to room temperature, the absorbance at 562 nm of each solution was recorded against a reagent blank prepared in 1.0 M sodium chloride. A standard curve was constructed from standards (5, 10, 25, 50, and 100 #g/ml) of bovine serum albumin (BSA) prepared in 1.0 M sodium chloride in order to obtain the concentration of protein in each of the experimental samples. The two-tailed Wilcoxon paired-sample test (Zar, 1984) was used to analyze the assay results.

RESULTS

Nonpolar Secondary Metabolitesfrom Aplysilla glacialis. Chemical investigation of A. glacialis collected from the mangrove lagoon at French Wells, Crooked Island, Bahamas, in 1988-1989 has resulted in the isolation and characterization of a variety of nonpolar secondary metabolites (Scheme 1 and 2). Different samples of A. glacialis were found to possess different mixtures of secondary metabolites (Table 1). Aplysilla glacialis (sample 88-275) collected in June 1988 was found to possess the labdane diterpene manorl (3), 5a,8aepidioxycholest-6-en-3/3-ol (4, "cholesterol endoperoxide"), and 5cr dioxy-24~-ethylcholest-6-en-3/3-ol (6, "24-ethylcholesterol endoperoxide"), along with a mixture of several other sterol endoperoxides that were not separated. Manorl (3) has been isolated from numerous terrestrial sources, but this is the first report of manorl from a marine source. Cholesterol endoperoxide (4)

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O

O

O 1

2

OH

H~S~176

3

4

NH il N/CH3

/N

I

H 5

SCHEMEI.

and 24-ethylcholesterol endoperoxide (6) have been reported from a variety of marine sources and appear to be especially common in sponges (Gunatilaka et al., 1981). A second collection of Aplysilla glacialis (sample 89-150B) was made in July 1989 from the same location in the Bahamas as the collection of the previous year. Separation of the dichloromethane-soluble portion of the crude methanol extract of the sponge yielded a sample of impure manorl (3), a mixture of sterol endoperoxides, and two diterpenes, spongia-16-one (7) and atisane-3/3,16a-diol (8), not found in the previous collection of A. glacialis. Spongia-16-one (7) has been reported recently from the dendroceratid sponge Dictyodendrilla cavernosa (Keman et al., 1990), while atisane-3/3,16ot-diol (8) had been isolated previously

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O

HO

% 7

6

OH

H O " " ~H ;

OAc c

....... '

8

9

CH3 HOOC ~

I NH +

HSO3-

"CH3

10 SCHEME2.

from the sponge Tedania ignis collected in the Caribbean Sea (Schmitz et ah, 1983). A third sample of Aplysilla glacialis (sample 89-150C) was collected from the same location in July 1989 and stored frozen for three months. The hexane extract of the lyophilized sponge tissue was separated to yield impure mano61 (3), a mixture of sterol endoperoxides, and pure spongia-15a, 16ot-diacetate (9). Spongia-15c~, 16ot-diacetate (9) had been reported previously from Spongia o~cinalis collected in the Mediterranean Sea (Cimino et al., 1982). A colloidal mixture of seawater and clear mucus from Aplysilla glacialis

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TABLE 1. CONCENTRATIONSOF NONPOLAR SECONDARY METABOLITES ISOLATED FROM DIFFERENT COLLECTIONS OF Aplysilla glacialis MADE AT FRENCH WELLS, CROOKED ISLAND, BAHAMAS

Collectiona Secondary metabolite Mano61 (3) Cholesterol endoperoxide (4) 24-Ethylcholesterol endoperoxide (5) Sterol endoperoxides Spongia-16-one (7) Atisane-3/3,16cx-diol (8) Spongia-15~,16a-diacetate (9)

June 1988 (88-275)

July 1989 (89-150B)

July t989 (89-150C)

0.039 0.061 0.062

0.26 " " 0.24 0.039 0.083 b

0.098 " c 0.093 b b 0.49

b b b

aValues are in percent dry weight. bMetabolite not detected during isolation procedure. CMetabolite not isolated in pure form.

was recovered from the plastic bags in which A. glacialis (sample 89-150C) was collected. Chemical investigation of this mucus yielded a mixture of sterol endoperoxides. TLC and [lH]NMR analyses were used to identify the mixture, but the GC-MS data were too complex to identify specific sterol endoperoxides from this mixture. Experiments were conducted in order to determine whether any of the nonpolar secondary metabolites isolated from the tissue of Aplysilla glacialis might be actively exuded directly into the water column. These experiments failed to detect the secondary metabolites isolated from A. glacialis in the water around the sponge. Fish Feeding Deterrents from Aplysilla glacialis. In situ fish feeding assays were carded out in the lagoon at French Wells, Crooked Island, Bahamas, in July 1989. The results (Figure 1) show that the pure secondary metabolites mano61 (3) and cholesterol endoperoxide (4) deter fish feeding by a natural assemblage of fish predators at a concentration of 1.6% dry weight (0.37% wet weight). Although these concentrations are approximately 10 times higher than was found in the sponge tissue, the dichloromethane-soluble portion of the crude methanol extract of A. glacialis (sample 89-150B) also was found to deter fish feeding when assayed at a natural volumetric concentration (Figure 1). Chemical investigation of the dichloromethane-soluble portion of the crude methanol extract ofA. glacialis (sample 89-150B) revealed that this collection contained a mixture of sterol endoperoxides, mano61 (3), spongia-16-one (7), and atisane-3/3,16~diol (8). Spongia-16-one (7) and atisane-3/3,16t~-diol (8) were unavailable for testing in their pure form.

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FIG. 1. In situ fish feeding deterrence assay results for mano61 (3, 1.6% dry weight), cholesterol endoperoxide (4, 1.6% dry weight), and the dichloromethane soluble portion of the crude methanol extract of Aplysilla glacialis ("natural concentration"--see text). Error bars indicate one standard deviation from the mean, n = number of experiments, P = significance value calculated using the two-tailed Wilcoxon paired-sample test.

Additional fish feeding assays were carried out in aquaria using a group of nine wrasses as the assay animals. Thalassoma lunare, an Indo-Pacific species, was used due to the unavailability of an endemic Caribbean species. The hexane, dichloromethane, and ethyl acetate extracts of Aplysilla glacialis (sample 89150C) were assayed at a concentration of 100 #g extract/mg krill, while the methanol extract was assayed at a concentration of 200/~g extract/mg krill. Each

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o f the crude extracts appeared to exhibit a deterrent effect on the feeding o f Thalassoma lunare at these concentrations, although the methanol extract was the only extract assayed in enough replicates to achieve favorable significance values (Table 2). Mano61 (3), spongia-15cc,16ct-diacetate (9), and a mixture o f sterol endoperoxides were isolated from the hexane extract o f this collection o f A. glacialis, while N , N ' - d i m e t h y l g l y c i n e bisulfite (10) and 1-methyladenine (5) were isolated from the methanol extract. The dichloromethane extract was determined by [1H]NMR to contain a mixture o f sterol endoperoxides and fatty acids, while the ethyl acetate extract appeared by [~H]NMR to contain mostly fatty acids. The pure secondary metabolites manor1 (3), cholesterol endoperoxide (4), and spongia-15ct,16c~-diacetate (9) were assayed at a concentration of 50 /zg metabolite/mg krill. None o f these pure compounds had an appreciable effect on the feeding of Thalassoma lunare (Table 2). N , N ' - D i m e t h y l g l y c i n e bisulfite (10) and 1-methyladenine (5) were not available for testing in this assay. Effect o f Secondary Metabolites of AplysiUa glacialis on Surface Fouling. A n in situ assay was carried out to determine the effect of mano61 (3) and cholesterol endoperoxide (4) on the fouling o f surfaces at a concentration o f 241 and 2 0 3 / z g / c m 2, respectively. Ten experiments were deployed for a period o f two weeks in southwest San Diego Bay in January 1990. After this period o f

TABLE 2. LABORATORYFISH FEEDINGASSAYRESULTSFOR MANOOL, CHOLESTEROL ENDOPEROXIDE, SPONGIA-15t2,16t~-DtACETATE,AND CRUDE EXTRACTSOF AplysiUa

glacialis ~

Sample Mano61 (3) Cholesterol endoperoxide (4) Spongia-15a, 16ctdiacetate (9)

Assay concentration (#g/ml)

Numberof replicates

Treated Control samples samples consumed consumed Significancevalue

50

7

6

7

0.975 < P < 0.99

50

12

7

12

0.10 < P < 0.25

50

11

8

9

0.975 < P < 0.99

100

8

3

8

0.10 < P < 0.25

100

7

3

7

0.10 < P < 0.25

100

4

0

4

0.10 < P < 0.25

200

10

1

8

0.025 < P < 0.05

A. glacialis hexane extract

A. glacialis CH2C12 extract

A. glacialis EtOAc extract

A. glacialis methanol extract

aAssay organism: Thalassoma lunare. Significance values were calculated using the G test of independence.

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BOBZINANDFAULKNER

time, visual inspection of the plates indicated that the surfaces were very sparsely settled by invertebrate larvae but were quite heavily colonized by bacteria, fungi, diatoms, and other unicellular algae. Therefore, the fouling of the plates was not assessed by visual inspection, but by a technique that would provide an index of the total biomass that had settled on the plates. The plates were analyzed for total adsorbed protein using the bicinchoninic acid (BCA) protein assay (Smith et al., 1985) to provide an indication of the total biomass that was present on each plate. The results indicate that mano61 (3) and, to a less significant extent, cholesterol endoperoxide (4) enhanced the rate of fouling when compared to the control plates (Figure 2). The results obtained for the experiments that used the crude extracts of AplysiUa glacialis were discarded due to the loss of material from the plates' surface upon immersion in seawater. The results for the experiments that were allowed to remain submerged for five weeks were also discarded due to uncertainties in the stability and solubility of the materials over this period of time.

Antimicrobial Activity of Secondary Metabolites and Crude Extracts of Aplysilla glacialis. Nine pure bacteria cultures were isolated from seawater samples collected in the lagoon at French Wells, Crooked Island, Bahamas, in July 1989. These seawater isolates were used for antimicrobial screening of the crude extracts and pure compounds from A. glacialis. The hexane, dichloromethane, ethyl acetate, and methanol extracts of Aplysilla glacialis (sample 89150C) were assayed against the nine bacterial isolates in the standard disk assay at a concentration of 250 /zg/disk. The hexane and dichloromethane extracts were inactive at this concentration against all nine of the bacterial isolates

FIO. 2. In situ fouling assay results for mano61 (3, 241 #g/cm2) and cholesterol endoperoxide (4, 203/zg/cm2). Assay conducted for two weeks in southwest San Diego Bay, Califomia, in January 1990. Error bars indicate one standard deviation from the mean, n = number of experiments, P = significance value calculated using the two-tailed Wilcoxon paired-sample test.

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screened. The ethyl acetate extract showed slight activity (8- to 10-mm zone of growth inhibition) against l,'ibrio sp. (strain H-46), Acinetobacter sp. (strain H60), and Flavobacterium sp. (strain H-73), while the methanol extract showed strong activity (17- to 21-mm zone of growth inhibition) against these three isolates in addition to slight activity against another l/ibrio sp. (strain H-12). The active, methanol extract ofAplysilla glacialis was separated using the activity against Flavobacterium sp. (strain H-73) to guide the isolation process. This yielded N,N'-dimethylglycine bisulfite (10) as an inactive component of the mixture and 1-methyladenine (5) as an active component. 1-Methyladenine inhibited the growth (ll-mm zone) of Flavobacterium sp. (strain H-73) at a concentration of 50/~g/disk.

DISCUSSION

Chemistry of Aplysilla glacialis. Chemical investigation of Aplysilla glacialis from French Wells, Crooked Island, Bahamas, revealed that different collections of the sponge contained different mixtures of nonpolar secondary metabolites (Table 1). Mano61 (3) and sterol endoperoxides were isolated from each of the three collections of A. glacialis made in June 1988 and July 1989, while the diterpenes, spongia-16-one (7), atisane-3/~, 16ot-diol ($), and spongia15a,16a-diacetate (9), were each isolated from only one of these collections. These differences may be due to the different procedures that were used in the storage, extraction, and isolation of each of the samples, although the high concentration of spongia- 15or, 16o~-diacetate in sample 89-150C (0.49 % dry weight) makes it appear unlikely that this compound could have gone undetected in the other collections. Alternatively, there may be a high degree of variability in the secondary metabolism of sponges in the French Wells population of A. glacialis or the species may be affected by unknown spatial, temporal, and environmental factors. The reports of several different spongian diterpene metabolites that were isolated from A. glacialis collected in British Columbia (Tischler and Anderson, 1989; Tischler et al., 1991) indicate that the secondary metabolism of A. glacialis does exhibit a high degree of variability. These compounds, exemplified by glaciolide (1) and cadlinolide A (2), would have been detected by the isolation procedure used in this work. Despite this, we believe that it would be premature to speculate as to which one of these possibilities has led to the observed differences in the chemistry of A. glacialis. We observed that Aplysilla glacialis exudes a mucus that is distributed as a thin film over the exterior of the sponge surface and that large quantities of this mucus are exuded when the sponge is handled or disturbed. The presence of sterol endoperoxides in the mucus of A. glacialis provides circumstantial evidence for the hypothesis that these compounds serve a defensive role. By

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distributing these compounds in the mucus, the sponge deploys them in a manner in which they could most effectively deter predation, overgrowth, or invasion by pathogens. The use of mucus exudates containing secondary metabolites is quite common in the defensive strategy of marine animals, especially among the opisthobranchs (Faulkner and Ghiselin, 1983). The secondary metabolites isolated from the mucus of the sponge Siphonodictyon coralliphagum were proposed to be responsible for the zones of dead coral polyps adjacent to this burrowing species (Sullivan, 1985). The isolation of sterol endoperoxides from the mucus of Aplysilla glacialis suggested that these compounds may also be actively exuded by the sponge into the sun'ounding water. The exudation of secondary metabolites by a sponge has been demonstrated in one case (Walker et al., 1985). This type of allelochemical defense strategy has also been observed in soft corals (Coil et al., 1982). The negative results from our field experiments do not eliminate the possibility that A. glacialis actively exudes its secondary metabolites into the surrounding environment, but it is apparent that when left undisturbed A. glacialis does not exude large quantities of compounds. Experiments with the sponge Aplysinafistularis indicated that this species exuded secondary metabolites at a rate of 10-3-10 -4 /~g/min/g dry weight of sponge (Walker et al., 1985). If A, glacialis exuded its metabolites at a similar rate, our experiments would have retrieved a quantity of metabolites well below the detection limits of TLC and [mH]NMR. Chemical Ecology ofAplysilla glacialis. Our field observations of Aplysilla glacialis indicated that this sponge was rarely eaten by fish, despite the fleshy, aspiculous nature of its tissue. This observation suggested that A. glacialis may be protected by a chemical defense provided by its secondary metabolites. There have been no reports in the literature of fish predation on dendroceratid sponges, but the absence of spicules in sponges of this order may be responsible for this observation because most of the studies of sponge predation by fishes have analyzed the gut contents of fishes for spicules in order to determine the species of sponges consumed (Randall and Hartman, 1968). Alternatively, the abundance of secondary metabolites that is common in dendroceratid sponges may provide this group with a very effective chemical defense against fish predation. The lagoon at French Wells, Crooked Island, Bahamas, is populated by yellowtail snappers (Ocyurus chrysurus), angelfishes (Pomacanthus spp. and Holacanthus spp.), grunts (unidentified species), bluehead wrasses (Thalassoma bifasciatum), foureye butterflyfishes (Chaetodon capistratus), juvenile barracudas (Sphyraena barracuda), and numerous schools of unidentified juvenile fishes. Of these fish species, only angelfishes are known to prefer sponges as a major component of their diet (Randall and Hartman, 1968), although butterflyfishes have also been identified as occasional spongivores (Dawson et al., 1955). Bluehead wrasses are omnivores that may include sponges in their diverse

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diet, while grunts, snappers, and barracudas are camivores that probably do not normally include sponges in their diet. The results from the in situ fish feeding assay conducted at French Wells show that the pure secondary metabolites mano61 (3) and cholesterol endoperoxide (4) deter fish feeding by a natural assemblage of fish predators at a concentration of 1.6% dry weight (0.37% wet weight) (Figure 1). This dry weight concentration is approximately an order of magnitude higher than that found in samples of Aplysilla glacialis, but the identification of sterol endoperoxides in the mucus of A. glacialis indicates that these compounds may be present at much higher concentrations at the sponge surface where they would be encountered by potential fish predators. This argument may not be applied to mano61 (3), but the high degree of significance of these data (Figure 1) indicates that this compound may be deterrent at lower concentrations. In support of this hypothesis, the dichloromethane-soluble portion of the crude methanol extract of A. glacialis (sample 89-150B), from which mano61 (3), spongia-16-one (7), atisane-3/3,16c~-diol (8), and sterol endoperoxides were isolated, was shown to deter fish feeding in situ when assayed at a natural volumetric concentration (Figure 1). Pure spongia-16-one (7) and atisane-3/3,16a-diol (8) were not tested due to lack of material. These results provide the first data on the effect of sponge extracts and pure secondary metabolites on fish feeding in situ at a location where the sponge species naturally occurs. The results from the laboratory fish feeding assay show that the crude methanol extract of Aplysilla glacialis (sample 89-150C) deterred feeding by Thalassoma lunare (Table 2). The other crude extracts also appear to deter feeding by T. lunare, but the limited number of replicates for these experiments resulted in poor significance values (Table 2). Mano61 (3), spongia-15o~,16adiacetate (9), and a mixture of sterol endoperoxides were isolated from the hexane extract of this collection of A. glacialis, while N,N'-dimethylglycine bisulfite (10) and 1-methyladenine (5) were isolated from the methanol extract. Pure samples of mano61 (3), cholesterol endoperoxide (4), and spongia-15c~, 16c~diacetate (9) did not have an appreciable effect on the feeding of Thalassoma lunare at a concentration of 50 tzg/mg (Table 2). N,N'-Dimethylglycine bisulfite (10) and 1-methyladenine (5) were not available for testing in this assay due to lack of material. The lack of a deterrent effect of the pure secondary metabolites may be an indication that these compounds must be present together, or present with some unknown component(s) of the crude extract, to elicit a strong negative response. Unfortunately, mixtures of the pure metabolites were unavailable for testing the hypothesis that the metabolites act synergistically to deter fish predation. We observed that Aplysilla glacialis was rarely overgrown by other invertebrates or algae and hypothesized that the secondary metabolites of A. glacialis

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may prevent fouling of the sponge's surface. The benthic environment in the tagoon at French Wells, Crooked Island, Bahamas, is a diverse and abundant habitat. The sea grass ThaUassia testudinum carpets the sand-sediment bottom wherever the depth is less than 15 ft. Several species of tunicates, hydroids, green algae, and brown algae are found at French Wells, while 23 species of sponges have been collected from this lagoon (Bobzin and Faulkner, unpublished data). Although many of these organisms co-occurred with A. glacialis on the mangrove prop roots, none of them were observed to overgrow this sponge. In fact, it was often found that A. glacialis would overgrow adjacent benthic species. An in situ assay was designed to determine the effect of mano61 (3), cholesterol endoperoxide (4)~ and crude extracts ofAplysilla glacialis on the fouling of surfaces. The results from the two week experiment carried out in San Diego Bay, California, indicate that mano61 (3) and, to a less significant extent, cholesterol endoperoxide (4) enhance the rate of fouling when compared to control plates (Figure 2). The results for the crude extracts ofA. glacialis were discarded due to the interference of components in these chemical mixtures with the BCA assay used to quantify the results and due to the inherent water solubility of these extracts, which allowed most of the plate coatings to be desorbed in a short period of time. The results for the five week experiment were also discarded due to the loss of compounds and extracts off of the plates after this period of time. These results indicate that mano61 (3) and cholesterol endoperoxide (4) most likely serve no role in the maintenance of the unfouled surface of Aplysilla glacialis. The observation that these metabolites actually enhance the rate of fouling may be simply due to the organic nature of the coatings that are applied to the experimental plates. The organic material may provide a nutritional source for bacteria and thereby increase the rate of colonization by bacteria, which is believed to be the first step in the succession of fouling (O'Neill and Wilcox, 1971). This point illuminates one of the shortcomings in the experimental design of the control plates. These plates should have possessed an equivalent organic coating similar to that of the experimental plates, thus providing the control plates with similar organic content and surface wettability characteristics. Despite these shortcomings, we believe that a truly deterrent metabolite would have overcome these factors. There have been few investigations into the antifouling role of marine secondary metabolites. Relevant in situ assays to determine the effect of secondary metabolites on fouling have been difficult to design (Bakus et al., 1983). Therefore, most investigations have employed pure cultures of a fouling species to determine the effect of secondary metabolites in laboratory experiments (Thompson et al., 1985; Rittschof et al., 1985; Targett et al., 1983). Our experimental design provides a quick and easy, albeit crude, method for deter-

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mining the effect that pure nonpolar secondary metabolites have on surface fouling in situ. Although we acknowledge that the experimental design requires some refinements, we found that the BCA assay technique was a very quick and accurate method for quantifying the results from such an experiment. The secondary metabolites produced by Aplysilla glacialis may also serve to protect the sponge from invasion by pathogenic marine microorganisms that the sponge filters out of the water while feeding. 1-Methyladenine (5) was identified as the antimicrobial constituent of A. glacialis (sample 89-150C) responsible for the growth inhibition of two strains of Vibrio sp., a Flavobacterium sp., and an Acinetobacter sp. These potentially pathogenic bacteria were isolated from seawater samples collected from within the lagoon at French Wells and would therefore presumably come into contact with A. glacialis in the natural environment. The antimicrobial activity of 1-methyladenine (5) may also be useful to A. glacialis as an aid to feeding by causing the agglutination of bacteria in the incurrent canals of the sponge (Bergquist and Bedford, 1978), in addition to a defense against invasion by pathogenic microorganisms. The field of sponge chemical ecology has received relatively little attention when one considers the amount of effort that has been invested in the isolation and characterization of the secondary metabolites produced by sponges. We have investigated several aspects of the chemical ecology of Aplysilla glacialis in an effort to determine whether the secondary metabolites produced by this sponge may serve ecologically relevant functions for the animal. Our results show that A. glacialis produces compounds, most notably mano61 (3) and cholesterol endoperoxide (4), that deter predation by fish in situ. A mixture of sterol endoperoxides, which are also believed to deter fish predation, were also isolated from the mucus which is exuded by A. glacialis. The distribution of these compounds in this mucus layer that coats the exterior of the sponge allows them to be deployed in a manner in which they could most effectively deter an attack by a fish predator. Although the results from the surface fouling experiments indicated that the compounds tested had no role in the maintenance of the unfouled surface of A. glacialis, the exudation of mucus may provide a physical means of continuously renewing the sponge surface, thereby sloughing off any growth that may start on the sponge. Another compound, 1-methyladenine (5), was found to inhibit the growth of several strains of marine bacteria, although the ecological purpose for this antimicrobial activity was not determined. It is quite apparent that A. glacialis employs a variety of strategies, both physical and chemical, to enhance its survival, and that the chemical aspect consists of more than one compound.

Acknowledgments--The authors would like to thank Ms. Mary Kay Harper, Mrs. Barbara Potts, Mr. Ron Iverson, and Dr. Denise Manker for help in collecting specimens and running assays. Ms. Karin Bj6rkman isolated and identified the marine bacteria strains used in our antimicrobial assaysand assisted in the constructionand deploymentof the foulingdeterrenceexperiments.

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Dr. Jon Clardy conducted the single-crystal X-ray diffraction analysis used to identify atisane3B,16oe-diol (8). Dr. Rita Colwell kindly provided us the opportunity to participate on research cruises in June 1988 and July 1989 aboard the R/V Columbus lselin (University of Miami). These cruises were funded by a research grant from the National Science Foundation (BSR-84-01397) to Dr. Rita Colwell, University of Maryland. This research was supported by grants from the California Sea Grant College Program (R/MP-46) and the National Science Foundation (CHE89-10821) to D.J.F.

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