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Journal of Chemical Ecology [joec] PP283-346658 October 18, 2001 15:31 Style file version Nov. 19th, 1999
Journal of Chemical Ecology, Vol. 27, No. 11, November 2001 ( c
°2001)
CHEMICAL DEFENSES OF THE SACOGLOSSAN MOLLUSK
Elysia rufescens AND ITS HOST ALGA Bryopsis sp.
MIKEL A. BECERRO,1,2,* GILLES GOETZ,1,3VALERIE J. PAUL,2
and PAUL J. SCHEUER1
1Department of Chemistry
University of Hawaii at Manoa
2545 The Mall, Honolulu, Hawaii 96822
2University of Guam Marine Laboratory, UOG Station
Mangilao, Guam 96923
(Received September 21, 2000; accepted July 15, 2001)
Abstract— Sacoglossans are a group of opisthobranch mollusks that have been
the source of numerous secondary metabolites; however, there are few exam-
ples where a defensive ecological role for these compounds has been demon-
strated experimentally. We investigated the deterrent properties of the sacoglos-
san Elysia rufescens and its food alga Bryopsis sp. against natural fish predators.
Bryopsis sp. produces kahalalide F, a major depsipeptide that is accumulated by
the sacoglossan and that shows in vitro cytotoxicity against several cancer cell
lines. Our data show that both Bryopsis sp. and Elysia rufescens are chemically
protected against fish predators, as indicated by the deterrent properties of their
extracts at naturally occurring concentrations. Following bioassay-guided frac-
tionation, we observed that the antipredatory compounds of Bryopsis sp. were
present in the butanol and chloroform fractions, both containing the depsipep-
tidekahalalideF.Antipredatory compounds of Elysia rufescens were exclusively
present in the dichloromethane fraction. Further bioassay-guided fractionation
led to the isolation of kahalalide F as the only compound responsible for the
deterrent properties of the sacoglossan. Our data show that kahalalide F protects
bothBryopsis sp. and Elysiarufescens from fish predation.This is the firstreport
of a diet-derived depsipeptide used as a chemical defense in a sacoglossan.
Key Words—Antipredatory role, herbivore–prey relationship, depsipeptides,
kahalalide F, sacoglossan mollusks, green algae, Elysia rufescens,Bryopsis sp.
∗To whom correspondence should be addressed at Center for Advanced Studies (CEAB, CSIC), Cami
de Sta Barbara s/n, E-17300 Blanes (GI), Spain. E-mail: mikel@ceab.csic.es
3Present address: Pharmacia, Searle Discovery Research, Mail Zone GG3D, 700 Chesterfield Parkway
North, Chesterfield, Missouri 63198.
2287
0098-0331/01/1100-2287$19.50/0 C
°2001 Plenum Publishing Corporation
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2288 BECERRO,GOETZ,PAUL,AND SCHEUER
INTRODUCTION
Sacoglossans (=ascoglossans) are a group of opisthobranch mollusks that feed
primarily on siphonaceous green algae (Williams and Walker, 1999). They are
highly specialized herbivores that can sequester functional chloroplasts from their
dietand use them as asource of photosynthetic energy(Clark et al., 1990; Williams
and Walker, 1999). Sacoglossans can also sequester secondary metabolites from
their diets or use the sequestered chloroplasts to convert bicarbonate into a
variety of carbohydrates that can be used for the synthesis of secondary
metabolites (Ireland and Scheuer, 1979; Ireland and Faulkner, 1981; Paul and
Van Alstyne, 1988; Gavagnin et al., 2000). It has been hypothesized that these
compounds defend sacoglossans against predators. These putative chemical de-
fenses are throught to provide ecological advantages and may function
as a driving force in the evolution of this group (Cimino and Ghiselin, 1998).
Althoughmarinenatural products chemistshaveisolatednumeroussecondary
metabolitesfrom sacoglossans(Faulkner,1992;Avila,1995),their ecologicalroles
remain largely uninvestigated. Many species synthesize polypropionates (Ireland
and Scheuer, 1979; Ireland and Faulkner, 1981; Ksebati and Schmitz, 1985; Dawe
and Wright, 1986; Roussis et al., 1990; Di Marzo et al., 1991; Vardaro et al., 1991;
Gavagnin et al., 1996). However, these compounds do not show an antipreda-
tory role when they have been experimentally tested (Hay et al., 1989; Roussis
et al., 1990). Both the chemically defended sacoglossan Cyerce nigricans and its
chemicallydefended hostalga Chlorodesmisfastigiatacontain chlorodesmin (Hay
et al., 1989). Although chlorodesmin significantly deters feeding by herbivorous
fishes (Paul, 1987; Wylie and Paul, 1988), it does not account for the antipredatory
properties of the mollusk (Hay et al., 1989). Two propionate-derived metabolites
isolated from the same mollusk species also lacked the deterrent properties of the
extracts (Roussis et al., 1990).
Our study focused on the sacoglossan Elysia rufescens and its food alga
Bryopsissp.Kahalalide Fis the majormetabolite of aseries of aminoand fatty acid-
deriveddepsipeptidesproducedby the greenalga Bryopsis sp. thatare accumalated
by Elysia spp. (Hamman and Scheuer, 1993; Hamman et al., 1996). Kahalalide
F shows a series of in vitro activities against tumor cell lines, viruses, and fungi
(Hamman and Scheuer, 1993), but no ecological roles have been investigated. In
our study, we investigated whether or not Bryopsis sp. and Elysia rufescens are
chemically defended against generalist fish predators and, if so, whether or not
kahalalide F is the compound responsible for the deterrent properties.
METHODS AND MATERIALS
Extraction and Isolation. We collected 5.4 kg wet mass of Elysia rufescens
and 4.67 kg wet mass of Bryopsis sp. by snorkeling at low tide near Black Point,
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ANTIPREDATORY ROLE OF KAHALALIDE F 2289
Oahu, Hawaii, during February 1995. Marilyn Dunlap and Alison Kay identified
the animals. A voucher specimen is deposited at the Bernice P. Bishop Museum,
Honolulu, BPBM 247679. Bryopsis sp. is an undescribed Bryopsis species highly
abundant in the reef flat at Black Point. In contrast to Bryopsis sp. at other sites
(Kan et al., 1999, personal observation), the specimens at Black Point lacked
macroepophytes. We used bioassay-guided fractionation to isolate and identify
ecologically active compounds. We used various solvents to extract and fraction-
ate the extracts from both species along gradients of polarity (Figures 1 and 2). The
general goal and approach was the same for the two species, but the specifics of the
process varied between E. rufecens and Bryopsis sp. The sacoglossans were ex-
tracted with ethanol (5 ×3 liters) and dichloromethane (CH2Cl2, 2 liters). Ethanol
and CH2Cl2extracts from E. rufescens were combined (282.3 g) and partitioned
between CH2Cl2and water (Figure 1). The aqueous layer was extracted with n-
BuOH, leaving 216.4 g of aqueous extract after evaporation. The n-BuOH layer
was combined with the CH2Cl2layer, concentrated (65.9 g), and partitioned be-
tween hexanes (30.5 g) and MeOH–H2O (9: 1) (35.4 g). The methanol layer was
collected and water was added to adjust the MeOH concentration to 60%. Extrac-
tion with CH2Cl2and concentration yielded 23 g of CH2Cl2extract and 12.4 g of
the aqueous methanol extract. The CH2Cl2fraction was subjected to ODS flash
column chromatography, by using a stepwise aqueous methanol gradient (50%
MeOH =ODS1, 70% MeOH =ODS2, 90% MeOH =ODS3, 100% MeOH =
ODS4, CHCl3–MeOH–H2O(7:3:0.5) =ODS5). Fraction ODS3 (12.2 g), was
subjected to ODS flash column chromatography by using a stepwise aqueous
acetonitrile gradient [50% MeCN, 60% MeCN, 70% MeCN, 80% MeCN, 100%
MeOH,CHCl3–MeOH–H2O (CMW,7:3:0.5)].The peptide-containing fractions
(monitored by TLC) were combined (8.3 g) and purified on an ODS column by
using a stepwise aqueous MeCN gradient solvent system (61%, 62%, 70% aque-
ous MeCN and 100% MeOH). The kahalalide F-containing fraction eluted with
61% MeCN (4.1 g), and it was passed through an ODS BondElut short column.
A reverse-phase HPLC separation (Ultracarb 10 ODS PO; MeOH–H2O–TFA,
65: 35:0.05) led to2gofpure kahalalide F. All the non-kahalalide F-containing
fractions and the HPLC side fractions were recombined to reconstitute what was
called fraction ODS6 (Figure 1).
The methanol (2 ×3 liters) and CHCl3–MeOH (1: 1) (2×3 liters) extracts
from Bryopsis sp. were combined (159.82 g) and partitioned between CHCl3and
water (Figure 2). The aqueous layer was extracted with n-BuOH (4.23 g). The
CHCl3layer was concentrated (11.09 g) and partitioned between hexanes (4.71 g)
and MeOH–H2O (9: 1) (6.87 g). The methanol layer was collected (2.67 g), and
waterwas addedto adjust theMeOH concentration to 60%. Extraction with CHCl3
and concentration yielded 4.2 g of chloroform fraction.
WeusedTLCand proton nuclearmagneticresonance (NMR spectrameasured
on a General Electric QE-300 or GN Omega 500 instrument) to detect kahalalide
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2290 BECERRO,GOETZ,PAUL,AND SCHEUER
FIG. 1. Scheme of the extraction and partitioning procedure used to obtain secondary
metabolites from the sacoglossan Elysia rufescens. Fractions enclosed in a double frame
were tested at their naturally occurring concentrations against natural fish predators in the
field. Fractions sharing the same double frame were tested in the same feeding experiment.
See text for more information on the isolation procedure and assay techniques.
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ANTIPREDATORY ROLE OF KAHALALIDE F 2291
FIG. 2. Scheme of the extraction procedure used to obtain secondary metabolites from
the green alga Bryopsis sp. Fractions sharing the same double frame were tested at their
naturally occurring concentrations against natural fish predators in the same field feeding
experiment.See text for more information on the extraction procedure and assay techniques.
F in the fractions and in the mucus produced by Elysia. The presence of secondary
metabolites in the mucus has been traditionally considered as indirect evidence
of the ecological role of the compound (Faulkner, 1992). To collect mucus, we
put slugs with seawater in zip-lock bags during collection and then transferred the
bags with slugs to containers with ice for transportation to the laboratory. Once
in the laboratory we took the slugs out of the seawater, which had taken on a
mucilaginous consistency due to the production of mucus by the slugs. We freeze-
dried the water–mucus solution, extracted with DCM, and then partitioned using
the Elysia scheme. Presence of kahalalide F was monitored in the fractions by
proton NMR analysis.
Antipredatory Experiments. We used field experiments to determine whether
Bryopsis sp. and Elysia rufescens are chemically defended against a natural coral
reef fish assemblage. Methods were similar to those of Becerro et al. (1998). We
added extracts from Bryopsis sp. and E. rufescens to an artificial diet consisting
of5gofground food, 2.5 g of carrageenan, and 80 ml of water. We used ground
material of the green alga Enteromorpha sp. or ground catfish pellets (Kruse’s
Perfection Brand) in an attempt to better mimic the nutritional characteristics
of Bryopsis sp. and E. rufescens in our artificial diets. We added the necessary
amount of extracts, fractions, or compounds relative to wet mass of the food to
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2292 BECERRO,GOETZ,PAUL,AND SCHEUER
match the natural concentration of extracts, fractions, or compounds relative to the
wet mass of Bryopsis or E. rufescens. The actual amount of extract (dissolved in
2 ml of dichloromethane–methanol 1: 1) added to the mixture varied according
to the percent yield (per wet mass) of the particular extract or fraction tested.
Control foods were prepared by adding 2 ml of solvent to the carrageenan-food
diet. The mixture was poured into 1-cm3molds containing a rubber O-ring, so
that safety pins could be used to attach cubes to ropes (40 cm long). Each rope
contained either four control or four treated food cubes. We placed 20 pairs of
control and treated ropes on the reef of Western Shoals, Apra Harbor, Guam. Pairs
were removed when approximately half of the cubes were eaten in any of the
treatments. We used Wilcoxon signed-ranks test for paired comparisons to test for
significant differences in the number of control and treatment cubes eaten.
RESULTS
In field experiments, extracts from Bryopsis sp. and Elysia rufescens sig-
nificantly deterred fish predators at naturally occurring concentrations. The an-
tipredatory properties of Bryopsis sp. are associated with the butanol and CHCl3
fractions (P<0.001 and P=0.01 respectively, Figure 3). TLC analysis showed
that kahalalide F is exclusively present in the active fractions.
The antipredatory properties of Elysia rufescens were associated with the
methylene chloride fraction (P=0.002, Figure 4), a combination of the butanol
and methylene chloride fractions from the first fractionation procedure (Figure 1).
Kahalalide F was the only compound responsible for the antipredatory properties
of the extract (P=0.02, Figure 5). Kahalalide F was also present in the mucus,
as observed by proton NMR, although the concentration at which it occured is
unknown.
DISCUSSION
Secondary chemistry seems to play a major role in the biology, ecology,
and evolution of mollusks. Sea hares, cephalaspideans, and nudibranchs either
sequester secondary metabolites from their diet or synthesize them de novo to
use them as chemical defenses (Faulkner, 1992; Avila, 1995). Although the data
available for sacoglossan mollusks seem to support a defensive role for these
compounds (Gavagnin et al., 1994a), experimental evidence is still scarce and
does not always support this hypothesis. Our study provides evidence that the
sacoglossanElysia rufescens andits hostalga Bryopsis sp.are chemicallydefended
against generalist fish predators. Kahalalide F, a major depsipeptide sequestered
by E. rufescens from the green alga Bryopsis sp., is the compound responsible for
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ANTIPREDATORY ROLE OF KAHALALIDE F 2293
FIG.3. Feeding deterrence of fractions from the greenalga Bryopsis sp. towards natural fish
consumersinthe field. Bars represent average(mean±1SE)number of control (empty bars)
and treated (filled bars) cubes eaten in the field by natural fish predators. Mean differences
between treatments and their respective controls were tested with Wilcoxon signed-ranks
testfor paired comparisons. The aqueous (water), butanol (BuOH),60% methanol (MeOH),
chloroform (CHCl3), and hexanes fractions were all tested at their naturally occurring
concentrations.
this activity. To our knowledge, this is the first report of a diet-derived depsipeptide
used as a chemical defense by sacoglossans.
Sequestration of secondary metabolites is widespread among opisthobranch
mollusks. There is ample evidence that sea hares and nudibranchs incorporate bi-
ologically active compounds in their tissues and use them for their own defense
(Faulkner, 1992; Avila, 1995). Similarly, sacoglossans also accumulate or mod-
ify secondary metabolites from their diets. Elysiella pusilla (=Elysia halimedae)
sequesters diterpenoids from its food alga Halimeda macroloba and uses them
as defense against fish predators (Paul and Van Alstyne, 1988). The sacoglossan
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2294 BECERRO,GOETZ,PAUL,AND SCHEUER
FIG. 4. Feeding deterrence of fractions from the sacoglossan Elysia rufescens towards
natural fish predators in the field. Bars and statistical values as in Figure 3. The aqueous
(water), 60% methanol (MeOH), methylene chloride (CH2Cl2), and hexanes fractions were
all tested at their naturally occurring concentrations.
Oxynoepanamensisfeeds on thechemicallydefended algaCaulerpasertularoides,
from which it incorporates caulerpin and caulerpicin (Doty and Aguilar-Santos,
1970). Chlorodesmin, a diterpenoid from the green alga Chlorodesmis fastigiata,
significantly deters feeding by some herbivorous fishes (Paul, 1987; Wylie and
Paul, 1988). However, the sacoglossan Cyerce nigricans specializes on feed-
ing on Chlorodesmis fastigiata (Hay et al., 1989), from which it incorporates
chlorodesmin. Although the sacoglossan is chemically defended, chlorodesmin
does not account for the deterrent properties of the mollusk (Hay et al., 1989).
Our data support a deterrent role for the metabolites ingested by Elysia rufescens
from Bryopsis sp. Similarly, the sacoglossan Costasiella ocellifera sequesters the
brominated compound avrainvilleol from its diet alga Avrainvillea longicaulis and
uses it as a defense against fish predators (Hay et al., 1990).
The production of polypropionate metabolites by marine mollusks is well
documented (Cimino and Sodano, 1993), including that by many sacoglossan
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ANTIPREDATORY ROLE OF KAHALALIDE F 2295
FIG. 5. Feeding deterrence of methylene chloride subfractions from the sacoglossan
Elysia rufescens towards natural fish predators in the field. Bars and statistical values
as in Figure 3. All subfractions were tested at their naturally occurring concentrations.
Subfraction labels: ODS1 =50% methanol, ODS2 =70% methanol, ODS6 =all com-
pounds(except kahalalide F) obtained fromthe 90% methanol subfraction, KF=kahalalide
F, ODS4 =100% methanol, ODS5 =chloroform–methanol–water (7:3:0.5), methylene
chloride (CH2Cl2), See text and Figure 1 for more information on the partitioning procedure
and isolation of kahalalide F).
species (Ireland and Scheuer, 1979; Ireland and Faulkner, 1981; Ksebati and
Schmitz, 1985; Dawe and Wright, 1986; Roussis et al., 1990; Di Marzo et al.,
1991; Vardaro et al., 1991; Gavagnin et al., 1996). Many polypropionates isolated
from mollusks have strong antibacterial (Biskupiak and Ireland, 1983; Dawe and
Wright,1986), cytotoxic(Ksebati and Schmitz, 1985), andichthyotoxic (Gavagnin
et al., 1994b; Vardaro et al., 1991) properties, and crude extracts from species con-
taining polypropionates significantly inhibit feeding by fish predators (Hay et al.,
1989). Polypropionates are also present in the mucus of sacoglossans (Di Marzo
et al., 1991; Vardaro et al., 1991). Deterrent secretions have been described in
many opisthobranchs including sacoglossans (see review by Avila, 1995), and the
presence of secondary metabolites in the mucus may be considered as indirect
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2296 BECERRO,GOETZ,PAUL,AND SCHEUER
evidence for the role of these compounds as a defensive mechanism against preda-
tors (Faulkner, 1992). The sacoglossan Elysiella pusilla (=Elysia halimedae) in-
corporates from its diet Halimeda macroloba the aldehyde halimedatetraacetate,
which the sacoglossan reduces to its corresponding alcohol and incorporates in
high concentrations in its body, mucus, and egg masses (Paul and Van Alstyne,
1988). The alcohol deters feeding by fish predators at natural concentrations (Paul
andVanAlstyne,1988). Polypropionatesmight functionsimilarly,although exper-
imental data on the sacoglossan Cyerce nigricans failed to support a deterrent role
for polypropionates (Hay et al., 1989; Roussis et al., 1990), and further research
is necessary to establish their role against predation. Whether polypropionates
play other biological or ecological roles is uncertain. There is evidence support-
ing the involvement of cyercenes in the regenerative processes after autotomy of
body parts in the sacoglossan Cyerce cristallina (Di Marzo et al., 1991), a process
widely distributed among sacoglossans that has received little attention (Lewin,
1970; Di Marzo et al., 1991; Trowbridge, 1994).
Our bioassay-guided fractionation procedure shows that kahalalide F is the
only compound responsible for the antipredatory properties of Elysia rufescens.
In the mollusk, kahalalide F is the major metabolite out of a group of several com-
pounds isolated from the mollusk and its dietary alga (kahalalides A–J) (Hamman
and Scheuer, 1993; Hamman et al., 1996; Goetz et al., 1997). Kahalalide F is found
in E. rufescens at concentrations between 0.4% (this study) to 1% (Hamann et al,
1996), which are several orders of magnitude higher than the concentration found
in the alga (0.0005%, this study). Kahalalide F shows remarkable clinical bioac-
tivity while the rest of the kahalalides lack significant cytotoxicity (Hamman et al.,
1996; Goetz et al., 1997). Although toxicity and deterrent activity are not neces-
sarily related (Pawlik et al., 1995), the diverse biological activity of kahalalide F
shows that the same compound may exhibit both clinically oriented and ecolog-
ically oriented activities. Since we only performed bioassay-guided fractionation
with the extracts from E. rufescens, the possibility that the minor, nontoxic kaha-
lalides may help deter predators in Bryopsis sp. cannot be completely ruled out.
DetailedTLC analyses of everyfraction showedthat allof the active algal fractions
contained kahalalide F, while we found no traces of kahalalide F in the nonactive
fractions.
It is worth noting that the concentration at which kahalalide F deters preda-
tors in the alga is very low (0.0005% of the algal wet mass). Even if all of the
kahalalides contribute to this effect, their total percentage of the algal biomass
is 0.0032%. Halimedatetraacetate, the major metabolite in Halimeda macroloba,
is about 0.2% of the algal wet mass (Paul and Van Alstyne, 1988). Both the
sacoglossan Cyerce nigricans and its host alga Chlorodesmis fastigata contain
the cytotoxic diterpenoid chlorodesmin (Hay et al., 1989). Although the sacoglos-
san is chemically defended against fish predators, chlorodesmin is not respon-
sible for the antipredatory properties of the crude extract when tested at natural
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ANTIPREDATORY ROLE OF KAHALALIDE F 2297
concentrations (less than 1% of the mollusk dry mass) (Hay et al., 1989). Two
pyrones from the same sacoglossan species accounted for 0.9% and 0.45% of the
dry mass of the mollusk and also failed to account for the repellent properties
of the crude extract (Roussis et al., 1990). Because of their low concentrations,
minor compounds may be easily overlooked in marine chemical ecology, yet they
may play a determinant role in the biology and ecology of benthic organisms. The
killer sponge Dysidea sp. is chemically defended against fish predators (Thacker
et al., 1998). However, the major compound, 7-olepupuane, does not account for
the total activity of the extract, suggesting that either addition or synergism of
other minor compounds enhances the activity of the major compound (Thacker
et al., 1998). Our study may be an example of how minor compounds (kahalalide
F, 0.0005% of the algal mass) account for the activities detected in the whole
extract.
Elysia rufescens is a highly cryptic but chemically defended species. In fact,
E. rufescens may have evolved a variety of defensive mechanisms to reduce the
chances of predation. We showed that Bryopsis sp. is a chemically defended alga,
that may provide the sacoglossan an associational refuge (Hay et al., 1990; Hay,
1992; Duffy and Hay, 1994). By feeding on Bryopsis sp., E. rufescens sequesters
algal chloroplasts and makes itself highly cryptic. However, predation may be
high on cryptic organisms (Trowbridge, 1994), so the acquisition of other defen-
sive strategies may expand the benefits of crypsis. E. rufescens sequesters the
antipredatory compound from Bryopsis, accumulates the compound up to several
times above the concentration in the alga, and becomes chemically defended it-
self. Moreover, E. rufescens releases the antipredatory compound into its mucus,
which may be considered as a defensive mechanism to deter predators (Lewin,
1970; Jensen, 1984; Trowbridge, 1994). Predation is an important factor influ-
encing mortality in benthic systems (Lubchenco and Gaines, 1981), and several
mechanisms may work together with the same goal. Predator–prey relationships
are important processes in marine benthic communities. Many of these relation-
ships are chemically mediated interactions between predators and their prey. By
investigating these associations, we will broaden our understanding of the biology
and ecology of benthic organisms and the factors that affect the evolution of these
predator–prey interactions.
Acknowledgments—We thank D. Galario, D. Ginsburg, C. Lacy, T. Mau, Y. Nakao, C. Nguyen,
J. Starmer, and W. Yoshida for their field and laboratory assistance and M. Dunlap and A. Kay for the
sacoglossan identification. We also thank staff members of The University of Guam Marine Laboratory
and The University of Hawaii Chemistry Department for providing not only their facilities but also
a nice environment in which to work. Comments from two anonymous reviewers greatly improved
this manuscript and are sincerely appreciated. This research was supported by a Basque Government
Postdoctoral Fellowship to M.A.B. and funded by NIH grant GM38624 to V.J.P. and Sea Grant College
Program and PharmaMar project to G.G. and P.J.S. This is contribution 454 of the University of Guam
Marine Laboratory.
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2298 BECERRO,GOETZ,PAUL,AND SCHEUER
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