Atapozoa Sp. and Its Nudibranch Predators Nembrotha Spp

MARINE ECOLOGY PROGRESS SERIES Vol. 59: 109-118, l990 Published January 1 l Mar. Ecol. Prog. Ser.

Chemical defenses of the tropical ascidian Atapozoa sp. and its nudibranch predators Nembrotha spp.

Valerie J. ~aul*,Niels ~indquist~,William ~enical~

' University of Guam Marine Laboratory, UOG Station. Mangilao, Guam 96923. USA 'Scripps Institution of Oceanography, University of California San Diego. La Jolla, California 92093-0228. USA

ABSTRACT The ascidian Atapozoa sp. is common in shallow reef habitats throughout the tropical Indo- Pacific. Collections from the central Philippine Islands, northern Sulawesi (Indonesia), Palau, Kwajalein. and Ant Atoll (near Ponape) were examined for the presence of secondary metabolites. In most cases, nudibranch molluscs Nembrotha spp. were found feeding on the ascidians. Like many dorid nudibranchs, these appear to obtain their defensive chem~calsthrough their diet. Both ascidians and nudibranchs contained large quantities of a serles of bipyrrole secondary metabolites previously described as the tambjamines. Concentrations of the different tambjamines and a related, highly blue- pigmented tetrapyrrole varied among collections of the ascidians and nudlbranchs Overall, concentrations of tambjamines and the tetrapyrrole were hlgher in the nudibranchs, and were the major components of the mucus exuded by Nembrotha spp. when irritated. Tambjamin? A and the tetrapyrrole were present in the nudibranchs but did not occur in detectable ~imountsin .4tapozoa.The ascidian and nudi.branch extracts and isolated metabolites were tested as feeding deterrents toward a variety of carnivorous fishes in field assays on 2 Guam reefs that differed in the types of fishes that fed during the assays. The crude extract, mixtures of tambjamines, tambjamine C, tarnbjamine F, and the tetrapyrrole were all significant feeding deterrents at or below natural concentrations. Tambjamines A and E were not deterrent when tested alone at natural concentrations; however, a I : 1 mixture of talnbjamines E and F was deterrent when tested below natural concentrations. Evidence is thus presented that both the ascidian and its nudibranch predators use the tambjarnines as chemical defenses agalnst predators.

INTRODUCTION Many benthic ascidians have been proposed to suffer relatively little predation by generalist predators (Mil- Ascidians are a rich source of bioactive secondary lar 1971, Goodbody & Gibson 1974, Stoecker 1980~). metabolites that show cytotoxic, antimicrobial, and High vanadium concentrations, low pH, and secondary antiviral activities (Ireland et al. 1988). Over 100 com- metabolites have all been proposed as defenses against pounds, primarily alkaloids or amino acid-derived predation and fouling in some species (Stoecker 1978, metabolites, have been isolated and chemically 1980a, b, c); however, Parry (1984) proposes that defined (Faulkner 1984, 1986, 1987, Fenical 1986, Ire- neither pH nor the presence of vanadium prevents land et al. 1988). Secondary metabolites from marine predation on ascidians. In the tropics, some angel- invertebrates have been proposed as chemical defen- fishes, spadefishes, filefishes, and pufferfishes are ses toward predators, pathogens, and fouling organ- known to consume relatively large amounts of asci- isms (Bakus 1981, Bakus et al. 1986, Co1.l & Sammarco dians (Randall & Hartman 1968, Myers 1983). Molluscs in press); however, few studies have experimentally such as Trochus niloticusand Turbo spp, have been tested the defensive functions of these compounds observed to feed on a variety of ascidians on the Great (Col1 et al. 1982, Thompson et al. 1982, 1985, Gerhart Barrier Reef (Parry 1984). Specialized predators on 1984, Bingham & Braithwaite 1986, La Barre et al. 1986, ascidians include n~olluscssuch as the lamellarians, Pawlik et al. 1986, 1987, 1988, Harvell et al. 1988, Paul cypraeids (cowries), and nudibranchs, and polyclad & Van Alstyne 1988, Wylie & Paul 1989). Feeding flatworms (Millar 1971, Morris et al. 1980, Parry 1984). deterrent functions of ascidian secondary metabolites On the other hand, some evidence for chemical defen- have not been examined. ses in ascidians has been observed. The larvae of the

O Inter-Research/Printed in F. R. Germany Mar. Ecol. Prog. Ser. 59: 109-118, 1990

ascidians Didemnum molle and Ectelnascidia turbinata MATERIALS AND METHODS were rejected by fish predators (Olson 1983, Young & Bingham 1987). Larvae of E. turblnata seemed to be Collections. Collections of Atapozoa sp, were made defended by low molecular weight organic met- in shallow water (usually < 10 m deep) habitats in abolites, although no deterrent secondary metabolites several areas of the western Pacific (Fig. 1). Ascidians were actually isolated (Young & Bingham 1987). were collected from Legan Island in Kwajalein Atoll, We commonly encountered the ascidian Atapozoa Ant Atoll near Ponape, Palau, Bunaken Island near sp, in shallow reef habitats throughout tropical areas of Manado in Sulawesi, and from several islands in the the western Pacific. The ascidians were abundant in central Philippines. The ascidians were most abun- the central Philippines, northern Sulawesi in Indonesia, dant in areas of high current or surge. After being Palau, Kwajalein Atoll, and Ant Atoll near Ponape collected, they were either frozen or immediately (Fig. 1). They were not encountered on reefs surround- placed in organic solvents (methanol andlor di- ing the islands of Ponape, Yap, Guam, or the northern chloromethane). Nudibranchs of the genus Nem- Mariana Islands. Atapozoa sp. was frequently grazed brotha, including N. crlstata, N. kubaryana and sev- by nembrothid nudibranchs, including Nembrotha cris- eral unidentified species, were commonly observed tata and N. kubaryana, but we did not observe preda- grazing on the ascidians. These nudibranchs were tion on the ascidian by generalist predators such as also collected and frozen or stored in acetone or fishes or molluscs. Preliminary chemical studies of the methanol. extracts of both ascidians and nudibranchs showed that Several collections were made of the exuded mucus they contained large amounts of nitrogenous secondary of Nembrotha spp. from the Philippines. Nudibranchs metabolites known as tambjamines (Carte & Faulkner were irritated by handling and the slimy blue-green 1983, 1986). The broad distribution of Atapozoa and its mucus that was exuded was collected on filter paper. close association with nudibranchs of the genus Nem- The mucus was dissolved in acetone, and the acetone- brotha allowed us to address questions about the chem- soluble extract was filtered and evaporated under ical defenses of these organisms: (1) Does Atapozoa reduced vacuum. produce secondary metabolites and do these com- Chemical extraction and analyses. Ascidians were pounds function as chemical defenses toward general- ground in a blender with acetone. The ground asci- ist predators? (2) How do the chemistry and feeding dians were filtered and extracted twice again with deterrent effects of the nudibranchs compare with the acetone, followed by extraction with methanol (3x), host ascidian? (3) How do concentrations of secondary and then the combined extracts were evaporated under metabolites vary among collections of the ascidian and vacuum leaving an oily organic residue. Nudibranchs nudibranchs? were soaked in acetone at least 3 times, and the

Mariana, Islands

rO0N Marshail 8 Guam (US) Islands ' APO I. Enewelako 8ik1n1a Yo p Atoll Atoll c - lsland.s , * AD Kwalaleln Palau D 1slands.r 0 -a Atoll " Truk C ' ( Olslands O . ,' B 0

I I

Fig 1 The western Paciflc indicating the collecting sites of Atapozoa sp and Nembrotha spp Collecting sites were. A, Legan Island on the leeward side of Kwajalein Atoll; B, Ant Atoll near Ponape; C, rock islands in Palau; D. Bunaken Island near Manado, Indonesia; E, Siguijor Island, Phihppines; F, Apo Island, Philippines; G, Sumilon Island, Phil~ppines;H. Sillirnan University Marine Lab, Dumaguete, Philippines Paul et al.: Chemical defense !S of an ascidian and its predators 111

acetone was evaporated under vacuum to leave an oily Dascyllus trimaculatus and Abudefduf spp. Thus, we extract. Lipid-soluble extract yields were calculated as tested the compounds toward 2 different populations of the dry weight of the extract divided by the dry weight predatory reef fishes since the species that fed from the of the extracted ascidian or nudibranch. ropes differed at the 2 sites. These fishes are common Identification of the secondary metabolites was throughout reef habitats of the western Pacific (Ames- accomplished by proton nuclear magnetic resonance bury & Myers 1982, Myers 1989) and represent a vari- (NMR) spectroscopy. Spectral values of the isolated ety of potential generalist predators of Atapozoa and metabolites were compared to published values in the Nem brotha. literature to identify known compounds. Several Thin strips of squid (ca 3mm thick, 1.5 cm X 1.5 cm metabolites were closely related to known metabolites square, dried with paper towels) were injected and but have not been previously reported. The structures coated with extracts or purified metabolites dissolved of these new tambjamines have recently been deter- in 1 : 1 diethyl ether: acetone. Extracts, isolated metabo- mined (Lindquist & Fenical unpubl.). lites, and mixtures of metabolites were tested at differ- Concentrations of the different tambjamines were ent concentrations representing the range of natural determined by isolating the metabolites by flash- concentrations found in Atapozoa and Nembrotha. column silica gel chromatography (using vacuum) Control squid strips were treated with equal volumes and reverse-phase high performance liquid chroma- of solvent only. When highly colored extracts or tography (HPLC). A solvent gradient from 100% di- metabolites such as the tetrapyrrole were tested, the chloromethane to 1 : 1 methanol : dichloromethane was control solvent was dyed with blue or green food col- used to initially fractionate the extracts by silica gel oring so that treated and control squid would visually flash chromatography. Tambjamines C, E, and F were match. Solvents were allowed to evaporate from both further purified with a second silica gel flash column by treated and control pieces before being offered to using a finer gradient of the same solvents. Final purifi- fishes. Since the extracts and isolated metabolites cation was obtained by C-18 reverse-phase HPLC with (with the exception of tambjamine aldehyde) were all a solvent mixture of 8:2 methano1:water with 09 M lipid-soluble, they adhere to the squid after the sol- ammonium acetate buffer. Tambjamine A was isolated vent evaporates and can then be placed in seawater. by passing a column fraction from the initial fractiona- Similar methods have been used for testing feeding tion of the crude extract of Nembrotha sp. through a deterrent effects of seaweed and invertebrate sec- Sephadex LH-20 column with 100 % methanol as the ondary metabolites (see Hay & Fenical 1988, Wylie & eluting solvent. The tetrapyrrole was obtained in large Paul in press). enough quantities for field assays by flash column Four treated and 4 control pieces of squid were chromatography of the combined fractions that pos- attached by paper clips to 2 separate yellow polypro- sessed this compound using hexane-ether solvent mix- pylene lines. These lines were attached to the reef as a tures. For some collections, proton NMR analysis of matched pair (0.25 to 0.5 m apart). Ten to 15 replicate fractions containing the tambjamines was used to esti- pairs were used in each trial, and each pair was mate the quantity of different tambjamines present by placed several meters apart on the reef. When at least integrating various peaks corresponding to different 4 pieces of squid were consumed from the pair, the tambjamines. numbers of control and treated pieces eaten were Feeding deterrent effects toward generalist car- recorded. Trials lasted only ca 15 to 20 min and com- nivores. Field bioassays were conducted at 5 to 8m pounds remained on the squid throughout the experi- depth at Fingers Reef in Apra Harbor and at Haps Reef ments as confirmed visually and by thin layer in Agat Bay, Guam, USA. Many herbivorous and car- chromatography (TLC) after the assays. It is possible nivorous fishes inhabit both reefs. The reef slope at that some material was lost in seawater during the Fingers is composed of scleractinian corals; Porites rus experiments; however, any losses of compounds is especially common. Fishes observed to feed during would make our results more conservative. The num- experiments at Fingers Reef included damselfishes bers of squid pieces eaten on control and treated ropes Abudefduf spp., wrasses Cheilinus fasciatus, Gom- were statistically compared by the Wilcoxon Signed- phosus varius, Halichoeres trimaculatus, Thalassoma Ranks Test for paired comparisons. Parametric statisti- lutescens, the emperor Lethrinus harak, and the trig- cal procedures were not appropriate for some of our gerfish Balistapus undulatus. Haps Reef faces the open assays because the differences among pairs in the ocean on the western side of Guam. The top of the number of control and treated squid pieces eaten were patch reef is composed of mostly dead Porites corals. not normally distributed. Since the Wilcoxon Test for Fishes observed to feed during experiments at Haps paired conlparisons is usually conservative when com- Reef included mostly triggerfishes Melichthys vidua, pared to the paired-sample t-test, we chose to use it Balistapus undulatus and occasionally damselfishes for all of the statistical analyses (Zar 1984). Table 1. Alapozoa sp. and Nembrotha spp. Secondary metabolite composition (PI: Philippines)

Dale Extract Total Tambjamine C Tambjamine E Tambjarnine F Tarnbjarnine A Tetrapyrrole Aldehyde yield ('X,) tambjamine (extract/dry yield ('5,) 'Yo %,dry ' dry '/o %,dry ')/L, %dry o ')/,dry ')h '%)dry mass x 100) (% dry mass) extract mass extract mass extract mass extracl mass extract mass extracl mass

Alapozoa sp. Mar 1087 5.8 1.80 28.8 1.68 2.7 0.16 0 0 0 0 0 0 0 0 Manado, lntlonesia Alapozoa sp Sep 1987 9.7 1.40 11.1 0.66 0 0 11.4 0.68 0 0 0 0 1.5 0.09 Pala1.1 A lapozoa sp. Mar 1988 2.4 0.16 1.0 0.02 0 0 0 0 0 0 0 0 6.1 0.14 Kwajdleln Aldpozoa sp. Sigu~jorIs., PI Alapozoa sp. May 1986 - Su~ililonIs., PI Aldpozoa sp. May 1987 - 0.89 - 0.07 S~lni~lonIs., PI Mar 1987 16.6 F. 10 25.0 4.08 0 0 0 0 3.1 0.5 3.1 0.5 6.3 1.02

N(!rnbrotha crislata Mar 1988 16.3 - Manatlo, lntlonesia Nmrbrolhd kltbaryand Mar 1987 17.5 2.20 6.3 1.11 0 0 0 0 5.1 0.89 trace amounts 1.3 0.22 Manddo, Intlonesia Mar 1987 6.9 0.67 7.9 0.55 0 0 0 0 0.9 0.06 0 9 0 06 0 0

Ner111,rolha sp. Sep 1987 18.0 ------major mctabolite Palau

N(~fr~brollidsp. Jun 1988 - 3.71 - 0.26 - 2.18 - 0.35 - 0.63 - 0.19 0.10 Sillillldn Un~v.Mrlr. Lab , PI

Nel~~brolhclS[)., c5xuded lrlucu .run 1988 - 65.0 9.8 - 48.8 - 16 - 0 0 4.8 - 0 0 Sill~~~~anUniv. Mar Lab , PI (% of acetone extract)

N~?~r~brollldk tlbdl ydncl, exuded May 1987 - 21.1 6.5 - 6.5 - 0 0 0 0 8.1 - 0 0

n111c.u~.Sun~ilon Is., PI (OX of acetone extract) Paul et al.. Chemical defenses of an ascidian and its predators 113

RESULTS tions contained quantifiable amounts of tambjamine A or the tetrapyrrole (Table l), although small amounts of Similar secondary metabolites were found in all col- the tetrapyrrole were often detectable by thin layer lections of Atapozoa sp.; however, the concentrations chromatography (TLC). of different metabolites varied among collections for Since some of the collections were handled differ- both the ascidian and the nudibranchs (Table 1). ently (frozen versus extracted immediately), and some Atapozoa sp. contained a series of bipyrroles known as tambjamines are unstable and may hydrolyze to tamb- the tambjamines (Fig. 2) that have been previously jamine aldehyde, small quantitative differences in the isolated from the bryozoan Sessibugula translucens, its concentrations of different tambjamines among collec- nudibranch predators Tan?bja a bdere and Tam bja tions may not be significant. Nonetheless, some pat- eliora, and the nudibranch Roboastra tigris that preys terns of distribution of the tambjamines are readily on Tambja spp. from the Gulf of California (Carte & apparent in Table 1. Overall, concentrations of crude Faulkner 1983, 1986). Tambjamines have also been organic extracts and total tambjamines were higher in found in the Indo-Pacific bryozoan Bugula dentata the nudibranchs Nembrotha spp. than in Atapozoa sp. (Lindquist & Fenical unpubl.). The tambjamines found Generally, the presence of the different tambjamines in collections of Atapozoa sp, included tambjamines C, in collections of Nembrotha resembled the patterns E, and F (Table 1; Fig. 2). Only tambjamine C has been found in Atapozoa from the same habitats. For exam- previously described (Carte & Faulkner 1983). None ple, collections of Atapozoa and Nembrotha from of the Atapozoa tambjamines were halogenated, Manado (Indonesia) and Kwajalein contained high although both bryozoans S. translucens and B, dentata amounts of tambjamine C but very low or undetect- contained halogenated tambjamines (Carte & Faulkner able concentrations of tambjamines E and F. Philippi- 1983, Lindquist & Fenical unpubl.). Some collections of nes collections of both organisms had high concen- Atapozoa also contained tambjamine aldehyde which trations of tambjamines C, E, and F (Table 1). In addi- has been shown to be a hydrolysis product of the other tion, extracts of all collections of Nembrotha contained tambjamines and therefore an artifact of isolation tambjamine A and the tetrapyrrole which were not (Carte & Faulkner 1983). None of the ascidian collec- detected in Atapozoa (Table 1; Fig. 2). Both metabolites have been previously described; tambjamine A was isolated from Sessibugula translucens (Carte & Faulkner 1983) and the tetrapyrrole was isolated from an undescribed Western Australian ascidian, the bryozoan Bugula dentata, and a mutant strain of the bacterium Serratia marcescens (Kazlaus- kas et al. 1982, Matsunaga et al. 1986). Tambjamine tambjarnine A R = H aldehyde was also present in some extracts of Nem- brotha, but this is a hydrolysis product and likely tarnbjarnlne C R = /)/ results from breakdown of the other tambjamines. Several collections of Atapozoa sp. and Nembrotha tarnbjarnlne E R = /\ spp. were made that were not quantified because they were extracted in the field; however, the tambjamines tarnbjarnlne F R = were identified and their relative concentrations deter- ,,p mined. Of the tambjamines isolated from Atapozoa sp. from Ant Atoll, tambjamine C accounted for 66.4 O/O of the total tambjamines, tambjamine E was 20.2%, tambjamine F was 2.2 %, and the aldehyde was 11.2 %. From Nembrotha cristata from Ant Atoll, tambjamine A accounted for 39 % of total tambjamines, tambjamine C tarnbjarnlne aldehyde was 39 %, tambjamine E was 5.5 %, the aldehyde was

OCH, OCH, 11 % , and the tetrapyrrole was 5.5 %. From Nembrotha \ kubaryana from Sumilon Island, Philippines, tamb-

jamine C accounted for 30.8 O/O of total tambjamines, tambjamine E was 30.8%, and the tetrapyrrole was 38.4 %. From Nembrotha sp. from Apo Islands, Philip- tetrapyrrole pines, tambjamine C accounted for 11.8% of total Fig. 2. Structures of the major metabolites isolated from collec- tambjamines, tambjamine E was 47 %, tambjamine F tions of Atapozoa sp. and Nembrotha spp. was 5.9%, and tambjamine aldehyde was 35.3%. 114 Mar Ecol. Prog. Ser.

These relative concentrations are similar to those Fig. 3). Natural concentration of the Atapozoa extract reported in Table 1 since Atapozoa contained tamb- from Kwajalein was 2.4 O/O of dry weight; however, jamines C, E, and F and Nembrotha spp. contained Nembrotha extracts occurred at much higher concen- very low or undetectable amounts of tambjamine F and trations of 6.9 % of dry weight (Table 1). relatively high concentrations of the tetrapyrrole. Results of field assays with isolated tambjamines The exuded mucus of Nembrotha contained high and mixtures of tambjamines on 2 Guam reefs are concentrations of the tambjamines as well as of the shown in Fig. 4. Tambjamine C was a major metabo- tetrapyrrole. When the nudibranchs were initially lite in most collections of Atapozoa and Nembrotha handled the exudate was dark blue, resembling the spp. (Table 1).It was a significant feeding deterrent on color of the isolated tetrapyrrole. This dark blue color both reefs at concentrations as low as 0.4 to 0.5 O/O dry was also released into seawater if the nudibranchs weight, which is within the range of natural concen- were prodded or attacked. The exudate changed to trations for Atapozoa and below natural concen- green upon continual handling. Tambjamines F and A, trations for Nembrotha. Tambjamine F was a signifi- which were present in the extracts of the whole nudi- cant feeding deterrent at Haps Reef at 0.6% dry branchs, were in low or undetectable concentrations in weight (p < 0.004. grazing reduced 71.7 % relative to the mucus. The tetrapyrrole was present in relatively controls) but not at 0.4 % dry weight (Fig. 4). Again, high concentrations in the exuded mucus. these concentrations fall within the range of natural The crude extract of Atapozoa from Ant Atoll was a concentrations for tambjamine F. Tambjamine E was significant feeding deterrent (p = 0.05) and reduced not a significant feeding deterrent on either reef at feeding by 24 '10 relative to controls at a concentration concentrations of up to 1.2% of dry weight, which is of 2 % dry weight, which is below natural concen- well above natural concentrations (Fig. 4). Tamb- trations (Fig. 3). Feeding deterrent effects of extracts of jamine A was not deterrent at 0.2 '10 of dry weight, and Atapozoa and Nembrotha kubaryana from Kwajalein the aldehyde was not deterrent at 1.0 O/O dry weight; were compared at equal concentrations of 1 % of squid both were tested within their range of natural concen- dry weight. The Nembrotha extract was more deterrent trations. The most deterrent metabolite that we tested

but results were not significant at the low concen- was the tetrapyrrole, which was effective at 0.1 O/O dry trations used in the assay (0.1 > p > 0.05, 2-tailed test; weight. This concentration was within the range that occurs naturally in the nudibranchs and is much lower than concentrations found in the mucus. All of the CRUDE ATAPOZOAVS mixtures of tambjamines C, E, and F were deterrent at EXTRACT NEMBROTHA 0.5 % dry weight and were as effective as pure tambjamine C. Mixtures of tambjamines E and F were deterrent; tambjamine E alone was not.

DISCUSSION

The tambjamines were effective chemical defenses against generalist carnivorous fishes in field assays on Guam. These secondary metabolites are produced by the ascidian Atapozoa sp. in a variety of habitats in the western Pacific, as well as by several species of bryozoans. In addition, several species of nudibranchs of the genus Nembrotha feed on Atapozoa and appear to obtaln the tambjamines from their diet in relatively Flg.3. Results of field assays with crude extracts of Atapozoa high concentrations. Thus, these compounds serve as and Nembrotha kubarycna expressed as % of squid pieces chemical defenses for the nudibranchs as well. These completely consumed (X 2 1 SE]. Left: Crude extract of observations agree with the feeding deterrent effects Atapozoa obtained from Ant Atoll collections and tested at 2";, of squid dry weight which is below natural concen- previously reported for tambjamines from the bryozoan trations. Open bar. control; cross-hatch: treated. kght Deter- Sessibugula translucens and its nembrothid nudl- rent effects of Atapozoa sp. and Nembrotha kubaryana from branch predators. Mixtures of halogenated and Kivajaiein Atoll compared at equal concentrations of l?:, dry nonhalogenated tambjamines isolated from the bryo- weight. Open bar. Atapozoa extract; cross-hatch: Nembrotha extract. (B: Assays conducted at Fingers Reef, Guam.) Results zoan and nudibranchs were deterrent toward the were analyzed by the W~lcoxonSigned-Ranks Test for paired California spotted kelpfish Gibbonsia elegans in comparisons (l-tailed test unl~ssotherwise noted) aquarium assays (Carte & Faulkner 1986) Paul et al Chemical defenses of an ascidian and its predators 115

TAMBJAMINE C TAMBJAMINE F TAMBJAMINE E

TAMBJ-A ALDEHYDE TETRAPYRROLE TAMBJ TAMBJ TAMBJ E& F MIX 1 MIX 2 1:l MIX

Fig 4 Results of field assays with isolated tambjamines and mixtures of tambjamines on 2 Guam reefs expressed as O/oof squid pieces completely consumed (X5 l SE) A Assays conducted at Haps Reef, B Assays conducted at Fingers Reef, open bars control squid, cross-hatch treated squid Concent~ationsreplesent ",D dry weight of squid Tambj Mix 1 = 2 1 tambjamine C tambjamine F Tambj Mlx 2 = 6 2 1 tambjamine E tamblamlne F tambjamine C Data were analyzed by the Wilcoxon Signed- Ranks Test for paired coinparisons (l-taded)

Based on our field assays, not all of the tambjamines The most deterrent metabolite that we tested was the were equally deterrent. Tamblaniine C wa? the most tetrapyrrole that occurs mainly in the nudibranchs. The deterrent and was effective at a concentration of 0.4 % tetrapyrrole is one of the major nietabolites found in the dry weight. Tambjamine C was a major metabolite in mucus exuded by Nembrotha spp. when they are dis- most collections of the ascidian and nudibranchs. turbed. The deep blue color of the tetrapyrrole may be Tambjamine F was deterrent at 0.6 % dry weight, but a warning to potential predators when it is released not at 0.4 %, and was found as a major metabolite in into seawater. Since we reduced the effect of color by some collections of Atapozoa. Tambjamine E was not dylng the control pieces of squid blue to match the deterrent even at the relatively high concentration of treated pieces, the deterrent results were based only on

1.2 Ol0 dry weight; however, 1: 1 mixtures of tambja- the taste of this metabolite coated on the squid. The mines E and F were deterrent at 0.5 % dry weight. In other metabolite found in Nembrotha spp. but not in fact, all of the mixtures of tambjaniines C, E, and F that Atapozoa sp. was tambjamine A. This metabolite was were tested were as effective as any single tambjamine not deterrent at 0.2 O/O dry weight; however, we did not tested alone. Most collections of Atapozoa contained a have enough of the isolated metabolite to test it at its mixture of tambjamines including the most deterrent higher natural concentrations of 0.5 to 0.8 O/O dry one, tambjamine C. Tambjamine aldehyde, which is weight. Based on our chemical analyses and the assay probably not a natural product, was not effectlve at comparing the deterrent effects of Atapozoa and Nem- 1.0 % dry weight. However, we had some problems brotha, it appears that the nudibranchs are better testing this metabolite because, unlike the other defended chemically. Not only do they contain higher nietabolites, it was partially water-soluble and may concentrations of the tambjamines, but they also con- have dissolved into seawater during the assays. tain more of the highly deterrent tetrapyrrole. 116 Mar. Ecol Prog. Ser. 59: 109-118, 1990

Our observations on the relationship between are that compounds cannot be evenly distributed Atapozoa sp. and its nudibranch predators support throughout the food and water-soluble extracts or certain hypotheses about the chemical adaptations metabolites cannot be tested in these assays. We tried of dorid nudibranchs. Faulkner & Ghiselin (1983) to minimize the first problem by using thin strips of hypothesized that the evolution of shell-less opistho- squid which maximized the surface-to-volume ratio of branch mollusks such as the dorid nudibranchs was the pieces and by both injecting and coating the correlated with the presence of defense mechanisms metabolites on the squid. In support of these methods, based on chemicals derived from food. Dietary-derived many nudibranchs have higher concentrations of chemicals seem to be a general method of defense for metabolites in the dorsal mantle (Thompson et al. many different opisthobranch molluscs including sea 1982, Pawlik et al. 1988) and exude metabolites when hares (Faulkner 1988, Hay & Fenical 1988), ascoglos- they are irritated; thus, concentrations of metabolites sans (Paul & Van Alstyne 1988), and nudibranchs (Ka- are not evenly distributed throughout the animals. ruso 1987, Faulkner 1988, Pawlik et al. 1988). In the Many sponges also have higher concentrations of sec- example we have described, the nudibranchs appear to ondary metabolites in the surface layers than in the either modify or selectively sequester some of the sec- internal portions (Paul unpubl.). The only metabolite ondary metabolites found in their host ascidian. Both we tested that dissolved in seawater was tambjamine the deterrent tetrapyrrole and tambjamine A are pre- aldehyde. The other compounds were lipophilic and sent in relatively high concentrations in the nudi- adhered to the squid. branchs but not in Atapozoa. Unlike many nudibranch It is interesting that identical or closely related tamb- predators, Nembrotha spp. are not aposematically col- jamines are produced by different phyla of inverte- ored. These nudibranchs are the same olive green color brates including bryozoans and ascidians. Nembrothid as Atapozoa sp. and are relatively d~fficultto see on nudibranchs seem to specialize on prey items that their host. contain these compounds. Tambja spp. feed on Ses- Although the chemistry of many nudibranchs has sibugula translucens in the Gulf of California (Carte & been investigated, and many are known to contaln Faulkner 1983, 1986), Nembrotha sp. feeds on Bugula high concentrations of secondary metabolites (see dentata in Palau and Ponape (pers. obs.), and N. cris- reviews by Faulkner & Ghiselin 1983, Faulkner 1984, tata, N. kubaryana, and Nembrotha sp. feed on Karuso 1987), few studies have actually investigated Atapozoa sp. Because the tetrapyrrole is produced by the defensive roles of these rnetabolites. In many cases, bacteria as well as bryozoa and ascidians, and is the diets of the nudibranchs are not known and can related to bacterial pigments such as prodigiosin (Kaz- only be guessed based upon similarities between the lauskas et al. 1982, Matsunaga et al. 1986), it has been chemicals found in the nudibranchs and in other inver- suggested that the compound may be biosynthesized tebrates such as sponges (Faulkner 1984). Only a few by bacteria associated with bryozoans or derived from studies have used potential natural predators to food sources such as prodigiosin-producing bacteria examine the effectiveness of nudibranch secondary (Matsunaga et al. 1986). metabolites as chemical defenses (Cimino et al. 1982, In conclusion, our data show that Atapozoa sp. and Thompson et al. 1982, Carte & Faulkner 1986, Pawlik et the associated predators Nembrotha spp. occur com- al. 1988). This study is the first to demonstrate the monly throughout the tropical western Pacific. These effectiveness of dietary-derived chemical defenses in a animals contain several related tambjamines and con- nudibranch that feeds on ascidians. centrations of these metabolites vary among collections Few other studies have linked secondary metabolites from different habitats. We did not analyze for indi- found in ascidians to those found in their predators. vidual or population variation within habitats; how- Marine prosobranch molluscs of the family Lamel- ever, it appears that most naturally occurring combina- lariidae are known to feed on ascidians (Millar 1971). tions of the tambjamines are effectjve feeding deter- The tropical species Lamellana sp. has been found to rents toward generalist predatory coral-reef fishes. contain secondary metabolites that are similar to those Tambjamines C as well as combinations of tambja- produced by the tropical ascidian Didemnum char- mines C, E, and F were all effective feeding deterrents taceum (Andersen et al. 1985, Lindquist et al. 1988). at concentrations below natural levels. Nembrotha spp. The field assays used in these studies have both contained high concentrations of the tetrapyrrole advantages and disadvantages when compared to which was the most deterrent metabolite we tested. To those utilizing an artificial agar or carrageenan-based the best of our knowledge, this study is the first to d~et(e.g. Harvell et al. 1988). A major advantage is demonstrate chemical similarities between an ascidian that many carnivorous fishes readily consume the and its specialist predators and to experimentally squid pieces in the field and will immediately attack evaluate and compare the chemical defenses of nudi- both treated and control pieces. Major disadvantages branchs and their host ascidian. - Paul et a1 Chenlical defenses of an ascid~anand its predators 117

Acknowledgements. This research resulted from financial Goodbody, I., Gibson, J. (1974) The b~ologyof Ascidia nigra support by the National Institutes of Health (GM 38624 to (Sav~gny)V Survival in populations settled at different V. J P) and the Natlonal Sclence Foundat~on(CHE86-20217 times of the year. Biol. Bull mar. biol. Lab., Woods Hole to W F and a predoctoral fellowsh~pto N.L.). \\;e thank Dr 146. 217-237 Fi-ancoise Monniot, National I\luseum of Natural History, Harvell, C D , Fenicill, W., Greene, C. H. (1988). Chemical Paris, France for identifying Atapozoa sp. and Clay Ciirlson for and structural defenses of Caribbean gorgonlans identifying the nudibranchs. We are grateful to J. Faulkner, S (Pseudopterogorg~aspp.) I. Development of an In situ Nelson, J Pawllk, K. Van Alstyne and 2 anonymous reviewers feeding assay. Mar. Ecol. Prog. Ser. 49 287-294 who commented on earlier drafts of this n~anusci-~pt,and to H Hay, M. E , Fenical, W. (1988). Marine plant-herbivore Sangei- for assistance with the figures. This is conti-~butionNo. interactions the ecology of chemical defense. Ann Rev. 274 of the University of Guam h,Iarine Laboratory Ecol Syst 19 111-145 Ireland, C M, Roll, D. M.. Molinslu, T F, McKee, T C , Zabrisk~e,T. M., Swersey, J C (1988) Uniqueness of the marine chemical environment. categories of marine LITERATURE CITED natural products from invertebrates. In: Fautin. D G (ed.) Biomedical importance of marine organisms. Mem Calif. Amesbury, S. S., h?yers, R. F (1982) Guide to the coastal Acad Sci 13: 41-57 resources of Guam: Vol 1 The fishes University of Guam Karuso, P (1987) Chemical ecology of the nudibi-anchs. In: Press, Mangilao Scheuer, P J (ed.)Bioorganic manne chemistry Springer- Andersen. R. J., Faulkner, D. J., Cun-heng, H., Van Duyne, Verlag, Berlin, p 31-60 G. D., Clardy, J. (1985). Metabolites of the marine proso- Kazlauskas, R., Mai-wood. J F., Murphy, P. T., Wells, R. J. branch Lamellaria sp. J Am. Chem Soc 107. 5492-5495 (1982) A blue pigment from a compound ascid~an.Aust. J. Bakus, G. J. (1981). Chemical defense mechanisms on the Chem 35: 215-217 Great Barrier Reef. Sclence 211: 497499 La Barre, S. C., Coll, J. C , Sammarco, P. W. (1986). Defensive Bakus, G. J , Targett, N. h?, Schulte, B. (1986). Chemical strategies of soft corals (Coelenterata. Octocorallia) of the ecology of manne organisms an overview. J chem. Ecol. Great Barrier Reef I1 The relationship between toxicity 12. 951-987 and feeding deterrence. Biol. Bull. mar biol Lab . Woods Bingham, B L, Braithwaite, L F (1986) Defense adaptations Hole 171- 565-576 of the dendrochirote holothurian Psolus chltonoldes Clark Lindquist, N. Fenical, W,Van Duyne, G. D , Clardy, J. (1988). J. exp. mar. Biol Ecol. 98. 311-322 New alkaloids of the lamellarin class from the marine Carte, B., Faulkner, D J (1983) Defensive metabolites froin ascidian D~demnum chartaceurn (Sluiter, 1909) J. org. three neinbrothid nudibranchs. J org. Chem. 48 2314-2318 Chem. 53. 45704574 Carte, B., Faulkner, D J. (1986).Role of secondary metabolltes Matsunaga, S., Fusetani, N., Hashimoto, K. (1986) Bioactive in feeding associations between a predatory nudibranch, marine metabolites. VIII. Isolation of an antimicrobial blue two grazing nudibranchs, and a bryozoan. J. cheni. Ecol. pigment from the bryozoan Bugula dentata. Experientia 12 795-804 42: 84 Cimino, G , De Rosa, S.,De Stefano, S.,Sodano, G. (1982) The hlilliir, R. H (1971). The b~ologyof ascidlans. Adv. mar. Biol. chemical defense of four Mediterranean nudibranchs 9. 1-100 Comp Biochem. Physiol. (B) 73: 471474 Morris, R H . Abbott, D. P., Haderlie, E. C (1980) Intertidal Coll, J. C.,LaBarre, S .Sammarco, P. W,Williams, W. T , Bakus, invertebrates of California Stanford University Press, G. J. (1982) Chemlcal defences in soft corals (Coelenterata. Stanford, p 177-226 Octocorallia) of the Great Barrier Reef: a study of compara- Myers, R. F (1983) The comparative ecology of the shallow- tlve toxicities Mar Ecol. Prog Ser 8: 271-278 water species of Canthlgaster (Family Tetraodontidae) of Coll, J. C., Sammarco, P \V (In press). The role of secondary Gudm M.S. thesis, Univ. of Guam metabolites in the chem~calecology of marine inverte- hlyers, R. F (1989) Micronesian reef fishes Coral Graphics, brates- a meeting ground for b~ologistsand chemists. Proc. Territory of Guam 6th Internat. Coral Reef Symposium Olson, R. A (1983) Ascidian-Prochloi-on symbios~s:the role of Faulkner, D J. (1984).Mar~ne natural products: metabolites of larval photoadaptations in midday larval release and sett- marlne invertebrates. Nat Product Rep. 1 551-598 lement. Biol Bull. mar. biol. Lab., Woods Hole 165: 211-240 Faulkner, D. J. (1986). Marine natural products Nat. Product Parry, D L. (l984) Chemical properties of the test of ascidians Rep. 3. 1-33 in relatlon to predation Mar. Ecol. Prog. Ser 17. 279-282 Faulkner, D J. (1987) Marine natural products. Nat. Product Paul, V. J., Van Alstyne, K L (1988).The use of ingested algal Rep 4: 539-576 diterpenoids by Elysla halimeda Macnae (Opisthobran- Faulkner, D J. (1988) Feeding deterrents in mollusks. In chia: Ascoglossa) as antipredator defenses. J. exp mar. Fautin, D. G. (ed.)Biomedical importance of marine organ- Biol. Ecol. 119: 15-29 isms. Mem. Calif. Acad. Sci 13: 29-36 Pawlik, J. R., Albizati, K. F., Faulkner, D. J. (1986).Evidence of Faulkner, D. J., Ghiselin, M. T (1983). Chemical defense and a defensive role for hmatulone, a novel triterpene from the evolutionary ecology of dorid nudlbranchs and some other intertidal limpet Collisella hmatula. Mar. Ecol. Prog. Ser. opisthobranch gastropods Mar Ecol. Prog. Ser. 13: 30. 251-260 295-301 Pawlik, J. R., Burch, M. T., Fenical. W. (1987). Patterns of Fen~cal,W. (1986). Marine alkaloids and related compounds. chemical defense anlong Caribbean gorgonian corals: a In: Pelletier, S. W. (ed.) Alkaloids. chemical and biolog~cal preliminary survey. J. exp. mar. Biol. Ecol. 108. 55-66 perspectives, Vo1.4. John Wiley & Sons, Inc., New York, Pawlik, J. R, Kernan, M. R., Mollnski, T. F., Harper, M. K., p. 275-330 Faulkner. D. J. (1988).Defensive chemicals of the Spanish Gerhart, D J. (1984). Prostagland~nA?. an agent of chemical dancer nudibranch, Hexabranchus sanyulneus, and ~ts defense in the Caribbean gorgonlan Plexaura homomalla. egg ribbons: macrolides derived from a sponge dlet. J, exp. Mar. Ecol Prog. Ser 19. 181-187 mar Biol. Ecol. 119- 99-109 118 Mar. Ecol. Prog. Ser. 59: 109-1 18, 1990

Randall, J. E., Hartman, W. D. (1968). Sponge feeding fishes of Thompson. J. E., Walker, R. P., Wratten, S. J., Faulkner, D. J. the West Indies Mar Biol. 1. 216-225 (1982) A chemical defense mechanism for the nudibranch Stoecker, D. (1978). Resistance of a tunlcate to fouling. Biol. Cadlina luteornarg~nata.Tetrahedron 38: 1865-1873 Bull. mar biol. Lab., Woods Hole 155: 615-626 Van Alstyne, K. L., Paul, V. J. (in press). The role of secondary Stoecker, D. (1980a). Distribution of acid and vanadium in metabolites in marine ecological interactions. Proc. 6th Rhopalaea birkelandi Tohoka. J. exp. mar. Biol. Ecol. 48: Interndt Coral Reet Syrnp. 277-281 Wylie, C. R., Paul, V J. (1989). Chemical defenses in three Stoecker. D. (1980b). Relationships between chemical defense species of Sinularia (Coelenterata, Alcyonacea): effects and ecology in benthic ascidians. Mar Ecol. Prog. Ser 3: against generalist predators and the butterflyfish Chaeto- 257-265 don unimaculatus Bloch (Perciformes). J. exp. mar. Biol. Stoecker, D. (1980~).Chemlcal defenses of ascidians against Eco~.129: 141-160 predators. Ecology 61: 1327-1334 Young, C. IM., Bingham, B. L. (1987). Chemical defense and Thornpson. J. E.,Walker, R P., Faulkner, D. J. (1985). Screen- aposematic coloration in larvae of the ascidian Ecteinas- ing and bioassays for biologically-active substances from cidia turblnata. Mar. Biol. 96: 539-544 forty marine sponges specles from San Diego, California. Zar. J. H. (1984). Biostatistical analysis. Prentice-Hall, Inc.. USA. Mar. Biol. 88: 11-21 Englewood Cliffs, p. 150-155

This article was submitted to the editor Manuscript first received: May 31, 1989 Revised verslon accepted: August 29, 1989