J. Exp. Mar. BioL Ecol., 151 (1991)227-243 © 1991 Elsevier Science Publishers B,V. All rights i'eserved 0022-0981/91/$03.50

227

JEMBE 01643

Diet-derived chemical defenses in the sea hare Stylocheilus longieauda (Quoy et Gaimard 1824)

Valerie J. Paul and Steven C. Pennings University of Guam Marine Laboratory, UOG Station, Mangilao, Guam, USA

(Received 31 January 1991; revision received 23 April 1991; accepted 6 May 1991)

Abstract: The sea hares (Opisthobranchia: Anaspidea)generally consume seaweeds that are chemically rich in secondary metabolites and concentrate these secondary metabolites in their digestive glands. These sequestered compounds are hypothesized to function in chemical defense against predators, although this hypothesis has rarely been experimentally evaluated. We investigated the feeding relationship between the specialist sea hare Styiocheilus longicauda (Quoy et Gaimard 1824) and its food, the filamentous cyanobacterium Micvocoleus lyngbyaceus (Kutz.) Crouan. Stylocheilus are found almost exclusively upon Microcoleus in the ~.d. In the laboratory, Stylocheilus strongly preferred Microcoleus over seven other diets. Individuals fed Microcoleus increased in mass 508% in I wk, whereas individuals fed five other diets grew much more slowly or lost mass. On Guam, Stylocheilus concentrates the secondary metabolites malyngamide A and malyngamide B from Microcoleus and converts malyngamide B into its acetate. In a variety of assays, whole extracts of Microcoleus, Stylocheilus, and isolated malyngamides at natural concentrations deterred feeding by natural populations of herbivorous and carnivorous fishes in two different reef habitats. Thus, as with other specialist opisthobranchs, S. iongicauda is adapted to feed on chemically defended foods, and it uses sequestered secondary metabolites for its own antipredator defenses.

Key words: Chemical defense; Microcoleus; Plant-herbivore interaction; Sea hare; Specialization; Stylocheilus longicauda

INTRODUCTION

Many marine opisthobranchs, including the dorid nudibranchs (Ordo Nudibranchia), the sea hares (Ordo Anaspidea), and the ascoglossans (Ordo Ascoglossa) sequester secondary metabolites from their diets (Faulkner & Ghiselin, 1983; Carefoot, 1987; Hay & Fenical, ~988). Dorid nudibranchs obtain metabolites from their invertebrate prey, primarily sponges but also ascidians, bryozoans, and coelenterates (Karuso, 1987, Faulkner, 1988, in press). Sea hares and ascoglossans obtain chemicals from their seaweed diets (Faulkner, 1984a, 1988, in press, Hay & Fenical, 1988). A great deal is known about the natural products chemistry of the opisthobranchs (Faulkner, 1984a,b, 1986, 1987, 1988; Karuso, 1987), however, much less is known about the natural functions of the sequestered compounds. Although it is widely assumed that sequestered

Correspondence address: V.J. Paul, University of Guam Marine Laboratory, UOG Station, Mangilao, GU 96923, USA.

Contribution 305 of the University of Guam Marine Laboratory.

228 V.J. PAUL AND S.C. PENNINGS

metabolites function in antipredator defense, this has rarely been shown. A few studies have shown a feeding deterrent effect toward predators for metabolites isolated from nudibranchs (Thompson et al., 1982; Carte & Faulkner, 1986; Pawlik et al., 1988; Paul et al., 1990a) or ascoglossans (Paul & Van Alstyne, 1988; Hay et ai., 1989, 1990). However, metabolites from the sea hares have generally not been tested in ecologically relevant assays (reviewed in Carefoot, 1987; Faulkner, in press).

Different species of sea hares prefer different algal diets. Some species, such as Aplysia californica Cooper, A. brasiliana Rang, and A. dactylomela Rang, are known to consume red algae, such as Laurencia spp. and Plocamium spp., and to concentrate the halogenated secondary metabolites from these algae. Other species, such as A. vaccaria Winkler and A. depilans Gmelin, consume brown seaweeds, such as Dictyota, and concentrate nonhalogenated terpenoids (Carefoot, 1987; Faulkner, in press). Pennings (1990a) investigated the factors affecting food choice for Aplysia californica. He found that A. californica of three size-classes grew faster on their preferred food Plocamium than on most other algal diets. Sea hares fed Plocamium sequestered a single monoter- pene from this diet; individuals fed Ulva lacked secondary metabolites. Sea hares fed Plocamium were ~so better defended from some predators than were individuals fed an Ulva diet. Thus, chemical defense may be one factor that contributes to the dietary choices of sea hares.

Stylocheilus longicauda (Quoy et Gaimard 1824) is the only sea hare known to preferentially feed on the filamentous blue-green alga (cyanobacterium) Lyngbya majuscula Gomont (= Microcoleus lyngbyaceus (Kutz.) Crouan) and "~ concentrate secondary metabolites from this cyanophyte. Some very toxic secondary metabolites, including aplysiatoxin and debromoaplysiatoxin, were reported from S. longicauda from Hawaii (Kato & Scheuer, 1974), although the natural function of these compounds is not known. These metabolites were likely of dietary origin since debromoaplysiatoxin was later isolated from several Lyngbya species on which numerous Stylocheilus were feeding (Mynderse et al., 1977; Cardellina et al., 1979a). The chemistry of Microcoleus is variable and numerous secondary metabolites have been isolated from different collections (Moore, 1981). The chemical variability of Microcoleus motivated us to conduct this study, because on Guam most collections contain malyngamides, compounds not previously reported from Stylocheilus.

In this study, we investigated the feeding preferences and chemical defenses of Stylocheilus longicauda on Guam. We first examined the food preferences and growth rates of Stylocheilus offered different seaweed diets. We then compared the secondary metabolite composition of Stylocheilus with its preferred food Microcoleus lyngbyaceus. Extracts and isolated secondary metabolites from the sea hares were tested as feeding deterrents toward natural populations of predatory fishes on two reefs on Guam. This is one of the first studies assessing the chemical defenses of sea hares toward natural predators.

CHEMICAL DEFENSES IN STYLOCHEILUS LONGICAUDA 229

STUDY ORGANISMS AND SITES

Stylocheilus longicauda is a small, diurnal, circumtropical sea hare in the family Notarchidae. This species is characterized by rapid growth, small maximum size, and a short life span. Field-collected animals rarely exceed 5 g in weight, although the animals may grow to 20 g in the laboratory (Switzer-Dunlap & Hadfield, 1979). In Hawaii, large aggregations of thousands of animals have been observed (Switzer-Dunlap & Hadfield, 1979; pers. obs). Switzer-Dualap & Hadfield (1977) reported that S. longicauda metamorphosed on Microcoleus and several species of red algae, but only Microcoleus supported rapid postlarval growth. In addition, large num- bers of juvenile S. longicauda could be found on Microcoleus in the field, suggesting that this cyanophyte might be a preferred natural settling site (Switzer-Dunlap & Hadfield, 1979).

For clarity and convenience, we refer to the cyanophyte Microcoleus lyngbyaceus as a seaweed or blue-green alga throughout this paper. Technically, this species is a filamentous cyanobacterium that forms large mats in many tropical habitats (see Humm & Wicks, 1980, for biology and taxonomy of the blue-green algae).

On Guam (13 °25' N, 144 ° 55' E), Stylocheilus longicauda were collected from patch reefs in Cocos Lagoon in 3-5 m of water. Animals were found exclusively on the cyanophyte Microcoleus lyngbyaceus and were very cryptic to a human observer on this food source. Generally, we collected large amounts of Microcoleus and brought it back to the laboratory to pick out the sea hares. We were able to collect Stylocheilus year-round as long as Microcoleus was available; however, Microcoleus abundance varied throughout the course of this study. We never collected Stylocheilus grazing on other algae in this habitat, although occasional individuals were found on highly epi- phytized algae or mixed algal collections from Apra Harbor, Guam.

We also collected ~ 100 specimens of S. longicauda in shallow water (1 m depth) near Black Point in Kahala Bay, Oahu, Hawaii, in July 1989. The animals were very abundant in this habitat, and were found on Microcoleus. The animals were placed in acetone, frozen, and returned to Guam for chemical analyses.

Experiments assessing the feeding deterrent effects of the Stylocheilus and Microcoleus extracts and isolated major metabolites of Stylocheilus toward reef fishes were carried out on two reefs on Guam, a Cocos Lagoon reef and Fingers Reef. Extracts of Microcoleus and Stylocheilus and the isolated metabolites were tested toward fishes on a patch reef in Cocos Lagoon where the Stylocheilus had been collected. This habitat had been previously used for similar experiments (Paul, 1987; Paul & Van A!styne, 1988) and contains a diverse assemblage of herbivorous and carnivorous fishes that graze during feeding assays, including Amblyglyphidodon curacao, Scarus sordidus, S. schlegeli, Acanthurus nigrofuscus, Naso lituratus, Zebrasoma flavescens, and Cheilinus fasciatus (names sensu Amesbury & Myers, 1982).

In addition, extracts of Microcoleus and Stylocheilus and extracts of Stylocheilus ink and egg masses were tested toward fishes on Fingers Reef in Apra Harbor, Guam.

230 V.J. PAUL AND S.C. PENNINGS

Carnivorous fishes are abundant in this habitat and species that fed during the assays included the wrasses Cheilinus fasciatus, Gomphosus varius, Thalassoma lutescens, and Epibulus insidiator, the triggerfish Balistapus undulatus, the emperor Lethrinus harak, and the sergeant majors Abudefduf spp. (names sensu Amesbury & Myers, 1982). This habitat had also previously been used for similar feeding experiments (Paul & Van Alstyne, 1988; Paul et al., 1990a).

METHODS

S T Y L O C H E I L U S FEEDING PREFERENCES

Experiments were conducted in laboratory aquaria to determine the dietary prefer- ences of Stylocheilus. Eight Stylocheilus were individually offered a choice between two species of algae that were weighted and placed at opposite ends of replicate 5-1 aquaria. The position of the two algae in each aquarium was determined randomly by flipping a coin. Individual sea hares were placed in the middle of each aquarium and their position (on alga A, alga B, or the tank) was noted hourly for 8 h. Replicates were not used if animals were not observed on algae at least three of eight times. Animals were recorded as preferring the algae upon which they were observed most (ties were dropped). A total of six choice tests were run: Microcoleus vs. Dictyota cervicornis (brown alga); Microcoleus vs. Enteromorpha clathrata (green alga); Microcoleus vs. Padina tenuis (brown alga); Microcoleus vs. the cyanophyte Schizothrix calcicola; Microcoleus vs. Schizothrix mexicana (tufted form); and Microcoleus vs. Schizothrix mexicana (prostrate form) (names sensu Tsuda & Wray, 1977). Individual Stylocheilus were used only once. Significance of preferences was assessed using a binomial test (two-tailed).

STYLOCHEIL US GROWTH

To determine the suitability of different species of seaweeds as food for Styiocheiius, we conducted laboratory growth experiments using six diets. Small (< 0.4 g) Stylocheilus were collected from Microcoleus and housed individually in 4-1 flow-through containers in a running seawater tank. Individuals (n - 2-7) were fed either Acanthophora spicifera, Boodlea composita, Enteromorpha clathrata, Herposiphonia sp., Padina tenuis, or Micro- coleus lyngbyaceus ad lib; uneaten portions were replaced with fresh algae daily (algal names sensu Tsuda & Wray, 1977). Mass of each Stylocheilus was measured before and after a 7-day period by blotting each animal dry and weighing it on an electronic balance (to nearest 10th of a mg). Data ( ~ change in mass) were analysed using ANOVA both with and without the initial mass of the Stylocheilus included as a covariate in the model. The covariate did not significantly improve the model, hence we report only the results of the analysis without the covariate.

CHEMICAL DEFENSES IN STYLOCHEILUS LONGICAUDA 231

CHEMICAL ANALYSIS

Collections of Microcoleus were extracted in 1:1 dichloromethane: methanol after grinding Microcoleus in a Waring blender. Solvents were evaporated by rotary evapora- tor under reduced pressure. Major lnetabolites were 1solated by vacuum flash silica gel column chromatography followed by silica gel high performance liquid chromatography (HPLC) in 60% ethyl acetate:40% hexane. Major metabolites were identified by proton nuclear magnetic resonance spectroscopy (NMR). Spectral values of the iso- lated metabolites were compared to published values in the literature to identify known compounds.

All collections of Stylocheih,.s were extracted three times with acetone. Whole animals were dropped into solvent and were not ground up. The acetone was evaporated to leave an oily residue. In addition to extractions of large numbers of animals, ~ 10 large Stylocheilus from Guam were extracted individually to examine individual variation in secondary chemistry. Egg masses of Stylocheilus were collected from the sides of aquaria and extracted in acetone after grinding in a Waring blender. Ink was collected by placing the animals on filter paper and poking them with forceps which made them release ink. The ink was dissolved off of the filter paper with acetone. Thus, only the organic-soluble component of the ink was collected. Major metabolites were isolated from Stylocheilus by silica gel column chromatography and HPLC as described above for Microcoleus.

FEEDING DETERRENCE ASSAYS

The susceptibility of live Stylocheilus to carnivorous fishes was observed at Fingers Reef in Apra Harbor, Guam. Ten individual Stylocheilus were tossed into the water column, and the fate of each animal (rejected or eaten) was noted. Similar susceptibility experiments were conducted at Waimea Beach Falls Park, Oahu, Hawaii, with 14

individuals collected in Hawaii. Deterrent effects of extracts and isolated metabolites were tested in Cocos Lagoon

and at Fingers Reef. Extracts and compounds were tested in several different types of artificial diets. Extracts from Microcoleus and Stylocheilus and the isolated metabolite malyngamide B were coated onto the palatable green alga Enteromorpha clathrata and tested toward herbivorous fishes in Cocos Lagoon. These methods have been previously described (Paul, 1987; Paul & Van Alstyne, 1988). Briefly, small pieces of Entercmorpha (,~ 250 mg wet mass) were coated with a solution of extract or pure compound dissolved in diethyl ether. Microcoleus extract was paired both with a solvent control and also with Stylocheilus extract at equal concentrations. Malyngamide B was paired with solvent

controls. Other assays were conducted by incorporating extracts and isolated metabolites into

artificial carrageenan-based diets. Harvell et al. (1988) described similar assays con- ducted in the Caribbean. For Fingers Reef assays, ground squid filet was stirred into a hot carrageenan mixture and allowed to gel. The recipe used was 2.5 g carrageenan, 2 g paraffin wax, 2 g ground squid (dry mass) and 60 ml water. Cubes made from this

232 V.J. PAUL AND S.C. PENNINGS

mixture were readily eaten by carnivorous fishes at Fingers Reef. For Cocos Lagoon assays, powdered fish chow pellets (3 g), rather than squid, were incorporated into the carrageenan to attract both herbivorous and carnivorous fishes. This second diet was designed to mimic the protein content and wet: dry mass ratio of seaweeds as closely as possible. For both diets, extracts or isolated metabolites dissolved in organic solvents or solvents only (for control diets) were stirred into the carrageenan mixture after it was heated. After the extracts or metabolites were added, the carrageenan diet was poured into molds and allowed to gel creating replicate l-cm 3 pieces. Black plastic o-rings were placed in each square in the mold before the food gelled. This allowed the food pieces to later be securely attached to safety pins on polypropylene lines.

For all of the assays, four pieces of food, either Enteromorpha or artificial diet cubes, were attached to polypropylene lines. Enteromorpha pieces were inserted into the lines; artificial diet cubes were attached by their o-tings to four safety pins on the lines. The ropes were placed on the reef in pairs (n -- 10-20) which usually consisted of one rope with treated pieces of food (extracts or isolated metabolites) and one rope with solvent controls. Pairs of ropes were removed from the reef when at least four of the eight pieces of food on the two paired ropes had been consumed. Results of the assays were scored as the number of food pieces completely consumed. Results were analysed using a two-tailed Wilcoxon signed ranks test for paired comparisons (Zar, 1984).

RESULTS

STYLOCHEILUS FEEDING PREFERENCES AND GROWTH

All individual So,locheilus preferred Microcoleus ovez all other seaweed choices, including other filamentous cyanobacteria such as Schi:othrix spp. (Table I).

TABLE I

Results of Stylocheilus feeding preference tests. P values were determined with a binomial test (two-tailed).

Alga Number of preferring P value

Microcoleus lyngbyaceus 7 0.016 Enteromorpha clathrata 0

Microcoleus 6 0.031 Dictyota cervicornis 0

Microcoleus 7 0.016 Padina tenuis 0

Microcoleus 7 0.016 Schizothrix calcicola 0

Microcoleus 8 0.008 Schizothrix mexicana (tufted) 0

Microcoleus 6 0.031 S. mexicana (prostrate) 0

C H E M I C A L D E F E N S E S I N STYLOCHEILUS LONGICAUDA 233

Growth of the Stylocheilus on six diets differed significantly (Fig. 1; F5,~8 = 10.20, p = 0.0001). Animals fed Microcoleus increased in mass 508 ~o in 1 wk. No other diet

650 t J3 to 550 ,<

Z 450 W 0 350 Z < "1- 250 (..) t-- Z 150 W 0 ~" 50 W a.

- 5 0 M E H P A B

DIET Fig. 1. Growth of Stylocheilus on six diets over a 7-day period. Data are ~ + 1 SE. ~ values that are not significantly different (ANOVA with Tukey comparisons, p < 0.05) are joined with a horizontal line. Abbreviations and sample sizes are: M, Microcoleus lyngbyaceus (n = 6); E, Enteromorpha clathrata (n = 7); H, Herposiphonia sp. (n = 2); P, Padina tenuis (n = 4); A, Acanthophora spicifera (n = 2); B, Boodlea composita

(n = 3).

yielded better than a 63~o increase in mass, and animals lost mass on diets of Acanthophora or Boodlea. Fecal pellets were observed in all containers, indicating that animals fed, at least to some extent, upon all diets.

C H E M I C A L A N A L Y S I S

Collections of Microcoleus from Cocos Lagoon contained malyngamides A and B as the major metabolites (Fig. 2). These metabolites have been previously reported as major metabolites of shallow-water varieties of Microcoleus from Oahu, Hawaii (Cardellina et al., 1978; 1979a,b; Moore, 1981). The combined malyngamides com- prised 50-60~o of the organic extract of Microcoleus, or ~ 2-3 ~o of the dry mass of the seaweed.

Stylocheilus from Guam contained malyngamides A and B, as well as a previously unreported compound malyngamide B acetate (Fig. 2). This compound was likely produced in the digestive glands of Stylocheilus from malyngamide B, since no traces of the acetate were ever found in Microcoleus. We were able to convert malyngamide B to the acetate by chemical acetylation with acetic anhydride in pyridine at room temperature. This synthetic metabolite was spectrally identical with the isolated compound from Stylocheilus, thus confirming its structure. Average organic extract yield of Stylocheilus from Cocos Lagoon was 24.4% + 9 .0~ SD of the animals' dry mass

234 V.J. PAUL AND S.C. PENNINGS

~OCH3

OCH 3 O O ~ l ~ l ~ ~

~H3 Cl OCH3

MALYNGAMIDE A

IOR

OCH 3 O O~'/N~

R=H MALYNGAMIDE B

R=COCH 3 MALYNGAMIDE B ACETATE

Fig. 2. Structures of major metabolites of Microcoleus and Stylocheilus.

(n = 7 collections). The malyngamides comprised ~ 30~o of the organic extract or ~77o of the animals' dry mass. The malyngamides were quantified by HPLC in individuals that were fed Microcoleus in the laboratory. In one individual, they com- prised 18 ~o of the animal's dry mass (60?/o ofthe organic extract). Tremendous variation was noted among individuals, and values for the combined malyngamides were as low as 1 To of the dry mass of some other individuals. In a combined extraction of numerous individuals collected at Cocos Lagoon, malyngamide A was 2 To, malyngamide B was 2.6~, and malyngamide B acetate was 2.2~ of the Stylocheilus dry mass.

Animals collected in Kahala Bay, Hawaii, contained only malyngamide A as the major metabolite (3.5 ?o dry mass). This is consistent with previous reports that Micro- coleus from Kahala Bay contained only malyngamide A, while other collections on Oahu contained both malyngamides A and B (Cardellina et al., 1978, 1979b).

Stylocheilus grown in laboratory tanks where Microcoleus was not available were eating primarily Enteromorpha. Based on chemical analyses by TLC and HPLC, these animals lacked malyngamides. This relationship between Stylocheilus secondary

CHEMICAL DEFENSES IN STYLOCHEILUS LONGICAUDA 235

chemistry and their diet has been previously reported for Stylocheilus raised in laboratory aquaria in Hawaii without Microcoleus. These animals lacked secondary metabolites (Mynderse et al., 1977). These data provide further evidence for a dietary origin for the malyngamides.

The malyngamides were found in the extracts of whole animals; however, neither the organic-soluble component of the ink or the egg masses contained measurable amounts of malyngamides. At this time, we do not know the secondary metabolite composition of the ink and the egg masses.

FEEDING DETERRENCE

At Fingers Reef, Stylocheilus offered to fish were mouthed and rejected by most fish. However, the wrasse, Thalassoma hardwickii (Bennett), did consume Stylocheilus after mouthing them several times. Of 10 individuals offered to fishes, four were eventually consumed by T. hardwickii. The other six were mouthed repeatedly by numerous fish, including T. hardwickii and Abudefduf spp., but were uneaten. Similar results were observed for the susceptibility trials conducted in the field in Hawaii. Of 14 individuals offered to fish, 10 were mouthed and rejected and four were eaten. Wrasses ate two Stylocheilus ', J pufferfish ate two. Stylocheilus were rejected by damselfish, wrasses, and some pufferfish. These observations suggest that, although Stylocheilus are protected to a large extent from predation, they can be consumed by some predators if encountered. However, to make these observations, we artificially increased encounter rates of predators with Stylocheilus by exposing the Stylocheilus in the water column. It is possible that sea hares offered to fish in the water column may be more susceptible to predation once encountered than if they were crawling along the bottom or on their host, Microcoleus.

Extracts of Microcoleus were significant feeding deterrents at natural concentrations to populations of reef fishes at both Fingers Reef (primarily carnivores) and Cocos Lagoon (primarily herbivores) (Fig. 3). At equal concentrations of 4 % dry mass, foods treated with Microcoleus extracts were significantly more deterrent toward fishes than foods treated with Stylocheilus extracts (Fig. 4). Although both extracts contained malyngamides as major secondary metabolites, Stylocheilus extracts contained relatively more fats and sterols (potential feeding attractants) and relatively less malyngamides than did Microcoleus extracts.

In three separate assays, Stylocheilus extracts were highly deterrent toward fishes at 10~ dry mass, ~ 1/2 of their natural concentrations (Fig. 5). They were not deterrent at 4~o dry mass, a concentration that was deterrent for Microcoleus extracts. Organic extracts of the ink and eggs were also highly deterrent toward carnivorous fishes at Fingers Reef at natural concentrations (Fig. 5).

The malyngamides were rarely significant feeding deterrents at concentrations of 1-2 ~o dry mass; however, malyngamide B and combinations of malyngamides A and B were deterrent at concentrations of > 2~o dry mass (Table II). Malyngamide B

236 V.J. PAUL AND S.C. PENNINGS

z 100 LU

h 80 W

,tn, l ._!

o 60

W

~ 40 Q.

b_ o

~" 20 z w

w ~- 0

Cocos 5/89

N=11 N=15

p=.05 p=.O04

Cocos 10/87

N=17

p=.O02 ,]"

Fingers 5/89

T

I---'1 CONTROL

EXTRACT

Fig. 3. Feeding deterrent effects of Microcoleus extracts at 4% dry mass toward herbivorous fishes at Cocos Lagoon and carnivorous fishes at Fingers Reef. Locations and dates of assays are shown at bottom of histogram. Vertical bars through each histogram show SE. N, number of paired comparisons used for each experiment. Significance values were determined by Wilcoxon signed-rank test for paired corr, parisons

(two-tailed).

acetate, which was found only in Stylocheilus and not Microcoleus, showed a non- significant trend toward being attractant at 1 and 2% (Table II). When the data from the two assays were combined to increase statistical power, malyngamide B acetate was

z I O0 N= 17 W ,,~ p=.02

h w 80 ,tn, _J

o 60

W

w 40 \ \ \ ~ . \ \ \

D.. \ \ \

o il,, ~" 20 \ \x Z \ \ \

%%% r'w ~ \ \ ILl o.. o ~N

N=17 p=.01

!

I I MICROCOLEUS

STYLOCHEII.US

Cocos Fingers

Fig. 4. Feeding deterrent effects of Microcoleus extracts compared with Stylocheilus extracts at equal concentrations of 4/% dry mass. Symbols and statistical tests are as in Fig. 3.

CHEMICAL DEFENSES IN STYLOCHEILU$ LONGICAUDA 237

I I CONTROL EXTRACT

z 100 W

h w 80

J

o 6 0

U') W

ul 40 11_ U. o

~ 20 z

bl n 0

N=15 N=14 N=19 N=21 N=12 N=11 p=.50 p=.01 p=.O01 p=.O02 p=.O03 p=.01

1.

Z . i ,

S 4% S I0%S 10%S 10% INK EGGS COCOS 5% 2%

Fig. 5. Feeding deterrent effects of Stylocheilus extracts. All assays were conducted at Fingers Reef except one noted as Cocos. S, Stylocheilus whole extract; ink, organic-soluble component of Stylocheilus ink; eggs, extract of Stylocheilus eggs. Numbers at bottom of histograms indicate concentrations of extracts as % dry

mass of artificial diet. Symbols and statistical tests are as in Fig. 3.

TABLE II

Results of field feeding deterrence assays at Cocos Lagoon with Microcoleus major metabolites. All assays used pellet food except where noted. Numbers in parentheses indicate sample sizes. +, attractant; - ,

deterrent.

Compound Concentration tested

1% 2% 2.5% 3%

Malyngamide A p = 0.66 (11) p = 0.41 (12)

Malyngamide A from Hawaii Microcoleus p = 0.72 (10)

Malyngamide B p = 0.09(12)+ p = 0.22(12)+

acetate

Malyngamide B p = 0.80 (7) p -- 0.64 (12)

Malyngamide B on Enteromorpha P = 0.55 (12) p = 0.004 (12)-

Malyngamide A and B combined P = 0.61 (12)

p = 0.02 ( 1 1 ) -

p = 0.02 ( i 9 ) -

238 V.J. PAUL AND S.C. PENNINGS

significantly attractant (two-tailed Wilcoxon test, n - 24, p = 0.024). The sea hares do not appear to convert malyngamide B into a more effective chemical defense.

There was no apparent synergistic effect of combined malyngamides as chemical defenses. To test this, we compared a combination of malyngamides A aal, d B with malyngamide B alone at Cocos Lagoon. At equal concentrations of 2% dry mass, 35.7 + 8.2 ~o of food cubes containing only malyngamide B were completely consumed, 60.7 + 6.8% of food cubes containing equal amounts of malyngamides A and B (1 each) were eaten (n = 14, p = 0.08, two-tailed Wilcoxon test). These data suggest that malyngamide B alone may be a better feeding deterrent, although the results were not significant.

DISCUSSION

This study demonstrates that Stylocheilus longicauda uses a diet-derived chemical defense against predators. As has been previously reported, Stylocheilus prefers to feed upon the filamentous cyanobacterium Microcoleus (Switzer-Dunlap & Hadfield, 1977, 1979), and it concentrates secondary metabolites from this food source (Mynderse et al., 1977). The chemistry of Microcoleus is highly variable; different collections yield very different secondary metabolites (Moore, 1981). Malyngamides A and B have been previously reported from Microcoleus but not Stylocheilus. Other secondary metabolites including aplysiatoxin, debromoaplysiatoxin, and stylocheilamide have been reported from Stylocheilus collected in Hawaii (Kato & Scheuer, 1974; Rose et al., 1978). Thus, Stylocheilus seems to accumulate whatever secondary metabolites are produced by the Microcoleus it consumes. Stylocheilus does not appear to store only a limited selection of the secondary metabolites present in its diet as has been proposed for other sea hares (Faulkner, in press). On Guam, the animals store the same major metabolites that are found in Microcoleus, and additionally convert malyngamide B to its acetate.

This is one of the first studies to experimentally demonstrate a defensive role for secondary metabolites acquired by sea hares. The chemical defenses were not entirely effective toward all predators, however, sin~:~e Thalassoma hardwickii and other wrasses sometimes consumed whole animals in field assays. Extracts and isolated malyngamides were deterrent at natural concentrations toward natural populations of reef fishes. In general, consistent results were obtained for the extracts and malyngamides even though a variety of artificial diets, including whole Enteromorpha and carrageenan-based diets, were used for the assays. We selected two different reef habitats because different predatory fishes were observed to feed on the two reefs. Thus, the extracts of Microcoleus and Styiocheilus and the malyngamides were broadly deterrent toward fishes. We have previously reported that Microcoleus extracts were deterrent in aquarium assays toward a variety of herbivorous fishes including Zebrasomaflavescens, Siganus argenteus, and Scarus sordidus (Wylie & Paul, 1988; Paul et al., 1990b; Paul, work in progress). Malyngamide A was deterrent toward Zebrasomaflavescens and Scarus sordidus (Wylie & Paul, 1988; Paul, work in progress).

CHEMICAL DEFENSES IN STYLOCHEILUS LONGICA UDA 239

The extracts of ink and eggs of Stylocheilus were also effective feeding deterrents toward herbivores, although these extracts did not contain malyngamides. The ink retained its purple color in the artificial diet, and it is possible that this coloration served as a warning to predators. However, we did notice that fish initially attacked both control and treated pieces of food in the field assays, so color alone was not deterrent. The eggs of some opisthobranchs contain diet-derived secondary metabolites (Paul & Van Alstyne, 1988; Pawlik et al., 1988); however, sea hare egg masses do not appear to contain the same natural products found in the adults (Faulkner, in press; Paul, pers. obs.). Sea hare egg masses have been reported to contain antimicrobial metabolites that can protect the eggs from bacteria (Kamiya et al., 1984). These were not the compounds responsible for deterrence in our assays because Kamiya et al. (1984) worked with water-soluble proteins and we worked with organic extracts. We still need to investigate the metabolites responsible for feeding deterrence of Stylocheilus egg masses.

Some fragmentary reports suggest that compounds from other sea hares deter preda- tors (e.g., Kinnel et al., 1979); however, these bioassays have generally not been con- ducted toward natural predators. Pennings (1990a) showed that Aplysia californica fed the chemically rich red alga Plocamium were better defended from some predators than were individuals fed an Ulva diet that lacked secondary metabolites. He did not, however, isolate and test the Aplysia secondary metabolites toward predators. Several isolated compounds from sea hares have been tested in field assays. Aplysin, reported from the sea hares Aplysia kurodai Baba and A. californica, was not deterrent toward herbivorous fishes in the Caribbean or Australia (Hay et al., lq87, 1988). Similarly the sea hare metabolite aplysistatin, reported from Aplysia angasi ( = dactylomela Rang) (Pettit et al., 1977), was not deterrent to herbivorous fishes on Guam (Paul et al., 1988). However, the Laurencia metabolite elatol, reported from Aplysia parvula Guilding (Faulkner, in press), was an effective herbivorous fish feeding deterrent in assays conducted in the Caribbean (Hay et al., 1987) and on Guam (Paul et al., 1988). Thus, ambiguous evidence had accumulated prior to this study to support the hypothesis that sea hare secondary metabolites are effective antipredator defenses.

The malyngamides were deterrent toward fishe~ only at relatively high concentrations of > 2 % dry mass. They are present in Microcoleus at this concentration and at higher concentrations in Stylocheilus. Other opisthobranch molluscs also sequester diet- derived chemicals defenses at higher concentrations than are found in their diets (Carte & Faulkner, 1986; Pawlik etal., 1988; Hay etal., in press). For example, the ascoglossan Elysia halimedae grazes on Halimeda and concentrates a modified tlalimeda diterpenoid at 7-8~o of its dry mass (Paul & Van Alstyne, 1988). The Halimeda diterpenoid is present at only ~ 1% dry mass in Halimeda. Similarly, the nudibranchs Nembrotha spp. concentrate diet-derived tambjamines at 2-6 % of their dry mass (Paul et al., 1990a). Tambjamines are present in their ascidian prey at only 0.5-2% dry mass. Opisthobranch molluscs seem to use high concentrations of secondary metabolites for effective chemical defense, possibly because they must compensate for their relatively high nutritional quality compared to their diets.

240 V.J. PAUL AND S.C. PENNINGS

Many opisthobranchs modify the secondary metabolites they obtain from their diets. This could occur: (1)to enhance the effectiveness of the chemical defense; (2)as a passive chemical transformation as the metabo!ite passes through the gut and digestive gland; or (3)as the first step of a detoxification mechanism. Several examples of chemical modification are known for the sea hares. Aplysia californica converts the Laurencia metabolite laurinterol to aplysin in the digestive gland (Stallard & Faulkner, 1974). Aplysin was not deterrent toward herbivores in field assays (Hay e~ al., 1987). Aplysistatin has been reported from the sea hare A. angasi as a likely oxidation product of the Laurencia metabolite palisadin A (Pettit et al., 1977). Aplysistatin was also not deterrent toward herbivorous fishes, although palisadin A was deterrent at similar concentrations (Paul etal., 1988). Gerwick & Whatley (1989) showed that A. dactylomela grazed on the tropical brown seaweed Stypopodium zonale and converted the seaweed metabolite 2-epitaondiol to 3-ketoepitaondiol. No comparisons were made of the bioactive properties of these two metabolites. In this study, Stylocheilus converted malyngamide B to its acetate. Malyngamide B acetate was not a feeding deterrent toward fishes; rather, it showed a trend toward being a feeding attractant. Thus, there exists no current evidence to suggest that sea hares enhance the biological activity of their dietary metabolites. Chemical conversion may occur as a byproduct of digestion or as a detoxification mechanism. Similarly, the chemical conversion of a major Halimeda metabolite that occurs in the ascoglossan Elysia halimedae does not appear to enhance its bioactivity toward predators (Paul & Van Alstyne, 1988).

In addition to providing chemical defenses to Stylocheilus, Microcoleus was the preferred food and supported rapid growth for this sea hare. Stylocheilus consistently selected Microcoleus over all other seaweeds in food choice experiments (Table I), and grew much better on Microcoleus than on any other seaweed (Fig. 1). Thus, Stylocheilus appears to be more specialized than many of the other sea hares that will consume several different seaweed diets (Carefoot, 1987). Because Stylocheilus prefers Micro- coleus despite its chemical variability, it is unlikely that Microcoleus secondary metabolites provide feeding cues. Compounds such as the nitrogenous malyngamides are structurally very different from the aplysiatoxins.

Multiple factors may promote specialization of the sea hare Stylocheilus for Micro- coleus. The sea hares grow fastest on Microcoleus and obtain effective chemical defenses from this diet. Additionally, the sea hares appear to be very cryptic on Microcoleus which may lower encounter rates with predators. Although we did not examine natural levels of predation on Stylocheilus, some herbivorous fishes tend to avoid feeding on Micro- coleus (Wylie & Paul, 1988; Paul et al., 1990b; Paul & Potter, in revision) suggesting that Stylocheilus would be rarely encountered and incidentally consumed by herbivorous fishes. Pennings (1990a) also showed that several factors including high growth rates and chemical defenses promoted narrow host range in the sea hare Aplysia californica. Additionally, small A. californica tended to be more specialized than larger animals (Pennings, 1990b). Since Stylocheilus is a small sea hare, size may play a factor in its high degree of specialization. Studies of the factors leading to specialization are central

CHEMICAL DEFENSES IN STYLOCHEILUS LONGICAUDA 241

to understanding plant-animal interactions. Hay & Fenical (1988) and Hay (in press) have discussed the importance of examining dietary specialization in marine herbivores, since fewer specialists appear to exist in marine systems. The results of this study support Hay's hypothesis that small, less mobile, herbivores often live on and graze chemically defended algae. The ability to chemically defend themselves from predators using diet-derived secondary metabolites may explain, at least in part, the specialized feeding behavior of Stylocheilus.

ACKNOWLEDGEMENTS

Financial support by the National Institutes of Health (GM 38624) is gratefully acknowledged. We thank P.J. Scheuer and R.E. Moore, University of Hawaii, for assistance in obtaining spectral data on the malyngamides. Acetylation of malyngamide B was accomplished in W. Fenicars laboratory (SIO) by V.J. Paul. C. Wylie, T. Nadeau, K. Meyer, and D. Carandang-Liberty assisted with many aspects of the field and laboratory research. J. Faulkner, W. Fenical, J. Pawlik, P.J. Scheuer, and an anonymous reviewer commented on earlier drafts of this manuscript.

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