Sequestration of Dietary Alkaloids by the Spongivorous Marine Mollusc Carsten Thomsa, Rainer Ebela, Ute Hentschelb, and Peter Prokscha,* a Institut für Pharmazeutische Biologie, Universität Düsseldorf, Universitätsstraße 1, Geb. 26.23, D-40225 Düsseldorf, Germany. Fax: 0049-211-81-11923. E-mail: [email protected] b Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany *Author for correspondence and reprint request Z. Naturforsch. 58c, 426Ð432 (2003); received January 15/February 4, 2003 Specimens of the spongivorous Mediterranean opisthobranch Tylodina perversa that had been collected while feeding on aerophoba were shown to sequester the brominated isoxazoline alkaloids of their prey. Alkaloids were stored in the hepatopancreas, mantle tis- sues, and egg masses in an organ-specific manner. Surprisingly, the known alkaloid aerothionin which is found only in A. cavernicola but not in A. aerophoba was also among the metabolites identified in wild caught specimens of T. perversa as well as in opisthobranchs with a documented feeding history on A. aerophoba. Mollusc derived aerothionin is pos- tulated to be derived from a previous feeding encounter with A. cavernicola as T. perversa was found to freely feed on both Aplysina in aquarium bioassays. The possible ecological significance of alkaloid sequestration by T. perversa is still unknown. Key words: Sponge-Opisthobranch Interactions, Marine Natural Products, Chemical Ecology

Introduction Pansini, 1997). The sibling A. cavernicola is pale yellow in appearance and dwells in under- Sponges of the genus Aplysina (syn. Verongia) water caves or at depths of up to 40 m and even harbor a wealth of structurally unique brominated below. Just like other Aplysina sponges Ð e.g. isoxazoline alkaloids (e.g. 1Ð4,Fig. 1) that are those occurring in the Caribbean sea (Pawlik et al., thought to be biogenetically derived from 3,5-di- 1995) Ð A. aerophoba and A. cavernicola are bromotyrosine (Tymiak and Rinehart, 1981). rarely attacked by predatory fish presumably due Whereas the spirocyclohexadienylisoxazoline moi- to the presence of the typical brominated Aplysina ety of most brominated alkaloids isolated from alkaloids. Nevertheless, the opisthobranch mollusc Aplysina sponges is structurally identical the re- Tylodina perversa is frequently found feeding on spective compounds differ by the nature of their A. aerophoba and is considered to be a specialized amine substituents linked to the carbonyl group predator on this sponge (Riedl, 1983) and perhaps adjacent to the isoxazoline ring (Fig. 1). Several also on A. cavernicola even though up to now it isoxazoline alkaloids from Aplysina sponges in- has not been reported from the latter from nature. cluding aerothionin (4), aerophobin-2 (2)orisofis- Morphologically defenseless opisthobranchs that tularin-3 (3)(Fig. 1) have recently been shown to are found in the oceans in a dazzling variety of act as strong feeding deterrents against marine shapes and colors are known to feed apparently fishes such as Blennius sphinx (Thoms, 2000) and unharmed on chemically defended sponges and are thought to play a crucial role in the chemical other marine invertebrates thereby accumulating defense of the sponges against predatory fish the chemical weaponry of their prey which in turn (Thoms et al., in preparation). is utilized for the chemical defense of the molluscs The Mediterranean sea hosts two Aplysina spe- e.g. against fishes (Faulkner and Ghiselin, 1983; cies: the sulphurous yellow A. aerophoba is a con- Paul and van Alstyne, 1988; Pawlik, 1988). T. per- spicuous sponge that lives exposed on hard bottom versa is no exception in this regard since it is like- substrata. It can be found at water depths as low wise morphologically defenseless (the rudimentary as 1 m and is among the most common sponges shell provides no efficient defense) and has been occurring in the Mediterranean sea (Riedl, 1983; shown to sequester brominated alkaloids from its

0939Ð5075/2003/0500Ð0426 $ 06.00 ” 2003 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com · D C. Thoms et al. · Sequestration of Alkaloids by the Mollusc Tylodina perversa 427

H CO 3 Br ence of T. perversa as judged by choice feeding

Br experiments and on the organ specific accumula- H N tion of dietary alkaloids during long term feeding HO O H NH N 2 of T. perversa on the sponges A. aerophoba and N N O A. cavernicola under controlled conditions in sea 1 H CO 3 Br water tanks. Furthermore we provide evidence Br based on electron microscopic studies that the se- H N questration of sponge metabolites by T. perversa H CO HO O H NH 3 Br N 2 N N is not paralleled by an accumulation of sponge- Br associated bacteria that are harbored in high den- O 2 OH Br sity in both A. aerophoba and A. cavernicola HO O H N O (Friedrich et al., 1999; Friedrich et al., 2001; N O O N Hentschel et al., 2001; Hentschel et al., 2002; Br N H CO O 3 Br 3 H OH Thoms et al., 2003). OH

Br Br O Materials and Methods Br HO O H OCH N N 3 N N H O OH Live specimens of the gastropod T. perversa O 4 were collected along with their prey sponge Aply- Br sina aerophoba in April 2002 off the coast of Br OCH Banyuls-sur-Mer, France. Specimens of the sponge OH O 3 OH OH were collected off the coast OH of Marseille, France. Three gastropods (group 1) N were immediately dissected into mantle, hepato- H N COOH OH 6 pancreas, alimentary duct, and reproductive or- 5 gans after collection. Six individuals were kept in Fig. 1. Structures of alkaloids: aplysinamisin-1 (1), aero- a sea water tank containing specimens of A. aero- phobin-2 (2), isofistularin-3 (3), aerothionin (4), uranid- ine (5), and A. cavernicola pigment (6) phoba and were allowed to feed ad libitum. After two weeks feeding on A. aerophoba under con- trolled conditions three gastropods were transfer- sponge prey (Teeyapant et al., 1993; Ebel et al., red to a tank containing specimens of the related 1999). sponge species A. cavernicola (group 3) and main- The majority of reports on the sequestration of tained there for two weeks (until the first signs of prey-derived secondary metabolites by opistho- decay at the prey sponge were observed) while the branch molluscs originates from analysis of speci- three remaining individuals were kept on A. aero- mens caught in the wild with a poorly documented phoba for another three weeks, yielding a total of feeding history (e.g. Faulkner and Ghiselin, 1983; five weeks (group 2). After these periods all gas- Pawlik et al., 1988). The full host range of prey tropods were dissected as described above. species amenable to respective opisthobranch spe- Egg masses that had been produced by molluscs cies, the organ-specific qualitative and quantitative of groups 2 and 3 during captivity and egg masses distribution of sequestered dietary secondary me- that had been removed from freshly collected gas- tabolites in the molluscs vs. those in their prey as tropods while feeding on A. aerophoba in the wild well as the metabolic fate of sequestered metabo- were lyophilized for subsequent extraction and lites are often unknown. Knowledge of the dif- HPLC analysis. Fresh egg ribbons were also pre- ferent factors that influence accumulation and re- served for electron microscopical analysis immedi- tention of putative defensive natural products, ately after collection as described below. however, is crucial for an assessment of the eco- Samples of the sponges (A. aerophoba and logical consequences of natural product sequestra- A. cavernicola) used for the feeding experiments tion by opisthobranch molluscs. In this study we were taken at the beginning and in the course of report on an investigation of the feeding prefer- the feeding experiments and analyzed by HPLC. 428 C. Thoms et al. · Sequestration of Alkaloids by the Mollusc Tylodina perversa

For HPLC analysis lyophilized samples of man- in 1:1 (v/v) propylene oxide/Epon 812 (Serva) the tle, hepatopancreas, and egg ribbons of the gastro- samples were embedded in Epon 812 at a temper- pods as well as lyophilized samples of the Aplysina ature of 60 ∞C. The embedded samples were sub- sponges were ground, extracted with methanol sequently sectioned with an ultramicrotome (OM and subsequently injected into an HPLC system U3, C. Reichert, Austria) and examined by trans- coupled to a photodiode-array detector (Dionex, mission electron microscopy (Zeiss EM 10, Zeiss, Germany). Routine detection was at 254 nm. The Germany). separation column (125 ¥ 4mmi.d.) was prefilled Feeding experiments with T. perversa employing with Eurosphere C-18 (5 µm) (Knauer, Germany). Aplysina and Axinella sponges were performed in Compounds were identified by their online UV six separate water tanks (size: 20 ¥ 20 ¥ 10 cm), spectra and by direct comparison with previously containing one specimen each of the sponges isolated standards (Ebel et al., 1997). Each com- Aplysina aerophoba, A. cavernicola and either pound in the samples analyzed was quantified Axinella damicornis or A. polypoides (all of them using calibration curves obtained for the respec- approximately 10 cm in diameter) and one individ- tive isolated natural products. ual of T. perversa. All the sponge species used for Electron microscopy was performed employing the experiment had a similar yellow color. The tissue samples of mantle, hepatopancreas and re- gastropod was placed at a starting point at about productive organs of gastropods of group 1 as well 15 cm equal distance from the three sponges. As as with freshly collected egg masses. The samples soon as it had selected one of the respective were cut into small slices with an ethanol-sterilized sponges the result was protocolled. For repetition scalpel, rinsed three times in sterile sea water, experiments the positions of the sponges in the fixed in 2.5% glutaraldehyde, and stored at 4∞ C. tank were altered randomly. They were then cut into smaller pieces of several mm3 in size, rinsed three times for 10 min in 1 ¥ Results and Discussion PBS and fixed overnight in 2% osmium tetroxide. After two additional rinses with 1 ¥ PBS, the HPLC separation of extracts of the two sponges pieces were dehydrated in an ethanol series (30%, A. aerophoba and A. cavernicola revealed the 50%, 70%, 100%) and incubated 3 ¥ 30 min in 1 ¥ same characteristic profiles of natural products as propylene oxide. Following overnight incubation previously described for both species (Teeyapant

Table I. Concentrations (µmol gÐ1 dry weight ð standard deviation) of alkaloids in various tissues derived from the opisthobranch Tylodina perversa (group 1: collected from the wild; group 2: after five weeks of feeding on A. aero- phoba; group 3: after two weeks of feeding on A. aerophoba followed by another two weeks of feeding on A. cavernicola) and in the prey sponges. Numbers of compounds refer to Fig. 1.

T. perversa mantle T. perversa hepatopancreas T. perversa egg masses Sponges Compound group 1 group 2 group 3 group 1 group 2 group 3 group 1 group 2 group 3 Aplysina Aplysina (n=3) (n=3) (n=3) (n=3) (n=3) (n=3) (n=3) (n=2) (n=1)* aerophoba cavernicola (n = 3) ( n = 1)**

Aplysinamisin-1 21.2 3.8 4.3 52.5 57.1 16.9 4.0 2.7 6.3 67.2 224.5 (1)(ð16.8) (ð2.2) (ð1.8) (ð15.0) (ð20.1) (ð13.2) (ð1.9) (ð0.1) (ð17.9) Aerophobin-2 (2) 8.2 6.6 4.8 21.6 25.7 3.8 11.9 11.7 16.1 29.2 3.6 (ð4.9) (ð3.7) (ð4.1) (ð2.6) (ð10.0) (ð1.9) (ð7.4) (ð3.7) (ð3.3) Isofistularin-3 (3) 0.0 0.3 0.2 15.1 27.0 0.2 0.1 0.1 0.0 18.5 2.4 (ð0.0) (ð0.4) (ð0.3) (ð1.9) (ð7.9) (ð0.1) (ð0.0) (ð0.1) (ð5.8) Aerothionin (4) 0.0 2.4 2.7 0.0 0.7 17.1 0.6 0.7 0.4 0.0 72.1 (ð0.0) (ð2.8) (ð1.5) (ð0.0) (ð0.6) (ð6.7) (ð0.5) (ð0.0) (ð0.0) A. cavernicola 0.0 0.0 18.4 0.0 0.0 45.6 0.0 0.0 11.5 0.0 246.4 pigment (6)(ð0.0) (ð0.0) (ð14.3) (ð0.0) (ð0.0) (ð14.8) (ð0.0) (ð0.0) (ð0.0)

*In the course of the aquarium feeding experiment with A. cavernicola only one egg ribbon was produced by the gastropods. ** The alkaloid pattern found in A. cavernicola tissue was similar to previous findings in earlier studies (e.g. Thoms et al., 2003). C. Thoms et al. · Sequestration of Alkaloids by the Mollusc Tylodina perversa 429 et al., 1993; Ebel et al., 1997; Ebel et al., 1999; small amounts of aerothionin (4) were unequivo- Thoms et al., 2003). Aplysinamisin-1 (1), aeropho- cally identified in the egg masses (Table I). bin-2 (2) and isofistularin-3 (3) were the predomi- Electron microscopy of mantle tissue, hepato- nating brominated alkaloids present in A. aero- pancreas, reproductive organs and egg ribbons de- phoba (Table I). No aerothionin (4) was detected rived from T. perversa revealed that no bacterial in extracts of A. aerophoba which is in congruence symbionts were present (data not shown) even with earlier studies (Teeyapant et al., 1993; Ebel though both A. aerophoba and A. cavernicola con- et al., 1997; Ebel et al., 1999). In addition, the tain large numbers of mostly extracellular bacteria chemically labile pigment uranidine (5) was also (Friedrich et al., 1999; Friedrich et al., 2001; present in A. aerophoba. Since uranidine is easily Hentschel et al., 2001; Hentschel et al., 2002; oxidized to black polymerous compounds it was Thoms et al., 2003). Thus sequestration of sponge not attempted to quantify the concentration of the alkaloids is apparently not paralleled by uptake of pigment in A. aerophoba unlike the concentra- bacteria from the sponge prey. tions of the chemically stable brominated alkaloids The hepatopancreas, mantle tissues as well as (Table I). A. cavernicola yielded aerothionin (4) egg masses of specimens of T. perversa from group and aplysinamisin-1 (1)asmajor brominated alka- 2 that had been feeding on A. aerophoba under loids. Isofistularin-3 (3) and aerophobin-2 (2) were controlled conditions for a total of five weeks all present as minor metabolites. In addition to the contained small but unequivocally detectable brominated alkaloids the pigment 3,4- amounts of aerothionin (4). The largest concentra- dihydroxyquinoline-2-carboxylic acid (6), which in tion of aerothionin (2.4 µmol gÐ1 dry wt.) was de- contrast to uranidine (5)isremarkably stable, was tected in the mantle, whereas the concentrations among the predominating secondary compounds of compound 4 in the hepatopancreas and in the identified in A. cavernicola (Table I). egg masses amounted only to 0.7 µmol gÐ1 dry wt., The alkaloid pattern and profile of the hepato- respectively (Table I). With the exception of aero- pancreas of specimens of T. perversa of group 1 thionin (4) the patterns of brominated isoxazoline that had been collected from the wild closely re- alkaloids (1Ð3) found in the hepatopancreas, in sembled that of their prey A. aerophoba. Aply- the mantle tissues and in the egg masses of the sinamisin-1 (1) was the major alkaloid in the hepa- gastropods from group 2 were similar to those of topancreas of T. perversa as well as in A. aerophoba T. perversa from group 1 (Table I). This chemical followed by aerophobin-2 (2) and isofistularin-3 similarity was most pronounced in case of the egg (3) which were present in similar concentrations masses produced by gastropods of groups 1 and in the hepatopancreas whereas compound 2 domi- 2 that both contained aerophobin-2 (2)asmajor nated over compound 3 in A. aerophoba (Table I). brominated alkaloid at almost identical concentra- In addition, trace amounts of uranidine (5) were tions (11.9 and 11.7 µmol gÐ1 dry wt., respec- likewise detected in the hepatopancreas thereby tively). confirming that the specimens of T. perversa col- The alkaloid pattern of gastropods from group lected from the wild while present on A. aero- 3 which had been feeding for two weeks on A. phoba had indeed been feeding on this sponge. cavernicola (after an initial three weeks feeding Interestingly, the mantle tissue of T. perversa from period on A. aerophoba) closely resembled that of group 1 yielded only aplysinamisin-1 (1) and aero- their last prey. This was again most striking for the phobin-2 (2)asbrominated alkaloids while in con- hepatopancreas which yielded large concentra- trast to previous studies (Ebel et al., 1999) neither tions of the A. cavernicola pigment (6) (45.6 µmol aerothionin (4) nor isofistularin-3 (3) was de- gÐ1 dry wt.) (Table I). Aerothionin (4) (17.1 µmol tected. gÐ1 dry wt.) and aplysinamisin-1 (1) (16.9 µmol gÐ1 Egg masses of T. perversa that had also been dry wt.) were present in almost the same concen- collected from the wild yielded a similar pattern trations whereas aerophobin-2 (2) and isofistu- of brominated alkaloids as found in mantle tissues larin-3 (3) constituted only minor secondary me- of the gastropods with aerophobin-2 (2) followed tabolites. The mantle tissues featured the pigment by aplysinamisin-1 (1)asmajor secondary constit- (6)asmajor nitrogenous constituent (18.4 µmol uents. In addition to compounds 1 and 2, however, gÐ1 dry wt.) followed by comparable concentra- 430 C. Thoms et al. · Sequestration of Alkaloids by the Mollusc Tylodina perversa tions of aerophobin-2 (2) (4.8 µmol gÐ1 dry wt.) Axinella spp. 8.3 % and aplysinamisin-1 (1) (4.3 µmol gÐ1 dry wt.). Egg masses that had been produced by T. perversa while feeding on A. cavernicola contained again aerophobin-2 (2) (16.1 µmol gÐ1 dry wt.) as major alkaloid followed by the pigment (6) (11.5 µmol gÐ1 dry wt.), aplysinamisin-1 (1) (6.3 µmol gÐ1 dry wt.) and aerothionin (4) (0.4 µmol gÐ1 dry wt.). A. cavernicola A. aerophoba 43.8 % 47.9 % It is particularly interesting to note that not only the pattern but also the concentrations of bromi- nated alkaloids present in all egg masses of T. per- versa analyzed in this study were remarkably similar irrespective of the large quantitative differ- ences of alkaloids found in A. aerophoba and A. cavernicola (Table I). Aerophobin-2 (2) consti- Fig. 2. Preferences of Tylodina perversa for selected tuted the major brominated alkaloid in all ana- sponges (in %) in choice feeding experiments. For each lyzed egg masses irrespective of the fact that aply- experiment one individual of T. perversa was offered one sinamisin-1 (1) (in both Aplysina species) and specimen of each of the sponge species, the total number of experiments was 48. aerothionin (4) (in A. cavernicola) clearly predom- inate over 2 in the sponges. The more lipophilic (based on comparison of reversed phase HPLC re- hypothesis 1: the presence of aerothionin traces tention times) alkaloids isofistularin-3 (3) and back to a previous feeding encounter with A. ca- aerothionin (4) were only present in small concen- vernicola;2:aerothionin is occasionally accumu- trations in the egg masses even though they are lated also by A. aerophoba; 3: aerothionin in conspicuous secondary metabolites in both Aply- T. perversa results from biotransformation of sina sponges. The chemical comparison of egg A. aerophoba alkaloids; 4: de novo biosynthesis of masses and to a lesser degree also of mantle tissue aerothionin by the gastropods. Hypothesis 1 of T. perversa with both Aplysina sponges reveals should involve feeding of T. perversa on A. caver- that sequestration of alkaloids by the gastropods nicola which up to now, however, has not been not merely mirrors the alkaloid profiles as present reported from nature. Therefore a feeding experi- in Aplysina sponges but appears to favor certain ment was conducted in this study involving speci- compounds such as aerophobin-2 (2). The reasons mens of T. perversa that were kept in tanks to- for the obvious differences in alkaloid profiles gether with A. aerophoba, A. cavernicola and the found in Aplysina sponges and e.g. egg masses of sponges Axinella damicornis and Axinella poly- T. perversa are unknown. poides.When given the choice between the dif- Previous studies on the presence of dietary alka- ferent sponges the gastropods clearly preferred loids in specimens of T. perversa that had been the Aplysina species (Fig. 2). Both A. aerophoba either collected in the wild while feeding on and A. cavernicola proved to be similarly attrac- A. aerophoba (Teeyapant et al., 1993) or that had tive to T. perversa based on the comparable num- been allowed to feed on this sponge species for bers of observation of the gastropods on the one week under controlled conditions (Ebel et al., sponges. This experiment as well as the observed 1999) correspondingly reported on the occurrence feeding of T. perversa on A. cavernicola over a of considerable amounts of aerothionin (4)inad- period of two weeks (group 3) unequivocally de- dition to the typical alkaloids from A. aerophoba monstrate that the gastropods freely feed on both (1Ð3). The presence of aerothionin (4)inthe opis- Mediterranean Aplysina sponges. The fact that un- thobranchs has been confirmed in this study (see like A. aerophoba no specimens of A. cavernicola results on alkaloid distribution in T. perversa from were observed at the site of collection of T. per- group 2 feeding on A. aerophoba under controlled versa at Banyuls-sur-Mer is not necessarily a valid conditions), but its origin still remains unclear. In argument against hypothesis 1 but probably re- general, four different hypotheses can be raised: flects differences in the ecology of both species. C. Thoms et al. · Sequestration of Alkaloids by the Mollusc Tylodina perversa 431

Whereas A. aerophoba grows exposed and can be perversa could be detected in the snails for at least found even at low water depths (around 1 m) up to four weeks after transferring B. perversa to A. cavernicola generally lives hidden in caves or at a parietin-free diet (Hesbacher et al., 1995). greater depths and is thus harder to find than the It is tempting to speculate on the possible eco- conspicuous A. aerophoba. logical roles of sequestered sponge alkaloids in Hypothesis 2 postulates at least occasional oc- T. perversa especially as it has been repeatedly currence of aerothionin in A. aerophoba.Whereas shown that sponge-derived natural products are this possibility can certainly not generally be ruled utilized by sponge-feeding gastropods such as out, it must nevertheless be clearly pointed out nudibranchs for their own chemical defense that all previous chemical studies on A. aerophoba against predators such as fishes (Cimino et al., and T. perversa that involved specimens from the 1993, Avila, 1995; Avila and Paul, 1997; Dumdei Canary islands (Teeyapant et al., 1993), the Medi- et al., 1997; Becerro et al., 1998). Recently it has terranean coast of Spain (Ebel et al., 1999) and been shown that brominated alkaloids (1Ð4)as France (Ebel et al., 1997) as well as from Croatia well as the A. cavernicola pigment (6) act as feed- (Teeyapant, 1994) unequivocally failed to detect ing deterrents against the Mediterranean fish aerothionin in the sponges. Hypothesis 3 which ex- Blennius sphinx (Thoms, 2000). However, the con- plains the origin of aerothionin through biotrans- centrations at which compounds 1Ð4 and 6 were formation of sequestered A. aerophoba alkaloids tested and at which they proved to be effective occurring in the gastropods would not only involve followed those that are present in A. aerophoba cleavage of sponge alkaloids such as 1 Ð 3 at the and A. cavernicola (see Table I). Since the alkaloid amide bond(s) but also re-assembly of the two concentrations in the mantle tissues and egg spirocyclohexadienylisoxazoline moieties obtained masses of T. perversa that are most vulnerable to upon cleavage and one putrescine unit thereby an attack by predators are considerably lower than giving rise to aerothionin. Compared to hypothe- found in Aplysina sponges it is difficult to simply ses 1 and 2 hypotheses 3 and 4 appear highly spec- transfer the results obtained in the previous study ulative with no experimental data available in sup- (Thoms, 2000) to our present investigation on port. Based on the presently available data T. perversa.The interesting question regarding the hypothesis 1 (previous feeding history of the gas- possible ecological advantage of alkaloid seques- tropods on A. cavernicola)isinour view the most tration for T. perversa can therefore presently not likely explanation for the origin of aerothionin in be answered but will have to await further studies. T. perversa, although this would involve a switch of host sponges by the gastropods. Acknowledgements However, if we assume that the presence of aerothionin in specimens of T. perversa from We gratefully acknowledge the technical assist- group 2 dates back from previous feeding on ance of the staff of the Laboratoire Arago at Ba- A. cavernicola, retention of this alkaloid (4) for nyuls-sur-Mer, France and the provision of Aply- clearly longer than five weeks and selective transfer sina cavernicola material by Prof. Jean Vacelet into the egg masses have to be postulated. In this (Centre d’Oce´anologie de Marseille, France). This context it is of interest to note that the anthraqui- work was supported by grants of the BMBF “Bio- none parietin which had been sequestered by spec- tecMarin” (to P. P. and U. H.) and by a grant of imens of the terrestrial lichen-feeding snail Balea the Fonds der Chemischen Industrie (to P. P.). 432 C. Thoms et al. · Sequestration of Alkaloids by the Mollusc Tylodina perversa

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