Journal of Chemical Ecology, Vol. 31, No.

7, July 2005 ( #2005)

DOI: 10.1007/s10886-005-5800-5

THE FATE OF Lyngbya majuscula TOXINS IN

THREE POTENTIAL CONSUMERS

ANGELA CAPPER,1,2,* IAN R. TIBBETTS,2 JUDITH M. O’NEIL,2,3

and GLENDON R. SHAW4
1
Smithsonian Marine Station, 701 Seaway Drive, Fort Pierce, FL 34949, USA
2
Centre for Marine Studies, School of Life Sciences, The University of Queensland,
Brisbane, QLD 4072, Australia
3
Centre for Environmental Science, University of Maryland, Cambridge, MD 21673, USA
4
National Centre for Environmental Toxicology (EnTox), University of Queensland,
Kessels Road, Coopers Plains, QLD, Australia

( Received October 8, 2004; revised January 14, 2005; accepted February 17, 2005 )

Abstract—Blooms of Lyngbya majuscula have been reported with increasing

frequency and severity in the last decade in Moreton Bay, Australia. A
number of grazers have been observed feeding upon this toxic cyanobacte-
rium. Differences in sequestration of toxic compounds from L. majuscula
were investigated in two anaspideans, Stylocheilus striatus, Bursatella leachii,
and the cephalaspidean Diniatys dentifer. Species fed a monospecific diet of
L. majuscula had different toxin distribution in their tissues and excretions. A
high concentration of lyngbyatoxin-a was observed in the body of S. striatus
(3.94 mg/kgj1) compared to bodily secretions (ink 0.12 mg/kgj1; fecal matter
0.56 mg/kgj1; eggs 0.05 mg/kgj1). In contrast, B. leachii secreted greater con-
centrations of lyngbyatoxin-a (ink 5.41 mg/kgj1; fecal matter 6.71 mg/kgj1)
than that stored in the body (2.24 mg/kgj1). The major internal repository of
lyngbyatoxin-a and debromoaplysiatoxin was the digestive gland for both
S. striatus (6.31 T 0.31 mg/kgj1 ) and B. leachii (156.39 T 46.92
mg/kgj1). D. dentifer showed high variability in the distribution of seques-
tered compounds. Lyngbyatoxin-a was detected in the digestive gland (3.56 T
3.56 mg/kgj1) but not in the head and foot, while debromoaplysiatoxin was
detected in the head and foot (133.73 T 129.82 mg/kgj1) but not in the
digestive gland. The concentrations of sequestered secondary metabolites in
these animals did not correspond to the concentrations found in L. majuscula
used as food for these experiments, suggesting it may have been from pre-
vious dietary exposure. Trophic transfer of debromoaplysiatoxin from
L. majuscula into S. striatus is well established; however, a lack of knowledge

* To whom correspondence should be addressed. E-mail: capper@sms.si.edu

1595
0098-0331/05/0700-1595/0 # 2005 Springer Science + Business Media, Inc.
1596 CAPPER ET AL.

exists for other grazers. The high levels of secondary metabolites observed in
both the anaspidean and the cephalapsidean species suggest that these toxins
may bioaccumulate through marine food chains.

Key WordsVStylocheilus striatus, Bursatella leachii, Diniatys dentifer,

lyngbyatoxin-a, debromoaplysiatoxin, bioaccumulation, Opisthobranchia, sec-
ondary metabolites.

INTRODUCTION

Opisthobranch mollusks are renowned for their ability to sequester a range of

dietary-derived compounds (Avila, 1995; Johnson and Willows, 1999).
Pennings and Paul (1993a) suggest that sea hares have a generic mechanism
for sequestering algal metabolites rather than mechanisms that are tightly linked
to different compounds. If this is the case, then the physiology of sequestration
would not be different among species. Study of the storage, secretion, and
egestion of these compounds by marine mollusks helps us to understand
defensive strategies and transfer of toxins within food webs.
Stylocheilus striatus Quoy and Gaimard 1832 (formerly longicauda;
Rudman, 1999) is an anaspidean species that can sequester, store, and transform
marine plant secondary metabolites (Faulkner, 1984; Avila, 1995), including
those found in the toxic cyanobacterium Lyngbya majuscula ( Watson and
Rayner, 1973; Kato and Scheuer, 1975; Pennings and Paul, 1993a,b; Pennings
et al., 1996). S. striatus adults preferentially feed and grow well upon a mono-
specific diet of L. majuscula (Paul and Pennings, 1991). Some L. majuscula
compounds (malyngamides and majusculamides) stimulate feeding by
S. striatus at low concentrations (Nagle et al., 1998; Nagle and Paul, 1999).
S. striatus sequester secondary metabolites in the digestive gland where trans-
formation to less harmful acetates could reduce negative postingestive conse-
quences ( Pennings et al., 1996). This hypothesis is supported by the isolation of
lyngbyatoxin-a and debromoaplysiatoxin acetates from S. striatus (Kato and
Scheuer, 1976). Both compounds are less toxic than their precursors in mice
bioassays (Kato and Scheuer, 1975; Gallimore et al., 2000).
The fate of L. majuscula secondary metabolites is known for S. striatus, but
other grazers of L. majuscula have received little attention. Cruz-Rivera and
Paul (2002) identified 43 invertebrate species in L. majuscula mats in Guam
including 14 species of opisthobranchs. The cephalaspidean Diniatys dentifer
(Adams, 1850) is one of the most abundant organisms observed in L. majuscula
blooms in Guam (Cruz-Rivera and Paul, unpublished data). D. dentifer was ob-
served feeding on L. majuscula, but this has not yet been quantified (Cruz-
Rivera, personal communication), nor its dietary repertoire established.
Bursatella leachii de Blainville, 1817, is a Bgeneralist^ grazer of cyanobacteria
THE FATE OF Lyngbya majuscula TOXINS IN THREE POTENTIAL CONSUMERS 1597

(Ramos et al., 1995) and is a common component of shallow water subtropical

fauna (Lowe and Turner, 1976; Paige, 1988). Like S. striatus, this anaspidean
species appears to use chemical cues from L. majuscula to induce settlement
and development of larvae (Switzer-Dunlap and Hadfield, 1977; Paige, 1988). To
date, there are no comprehensive studies to determine the fate of L. majuscula
compounds in D. dentifer and B. leachii.
In this study, we examined intraspecific differences in internal partitioning
of lyngbyatoxin-a and debromoaplysiatoxin between two populations of
S. striatus: (1) those previously feeding on L. majuscula; and (2) those naı̈ve
to L. majuscula. Interspecific differences in internal compartmentalization, sec-
retion, and egestion of lyngbyatoxin-a were investigated between S. striatus,
B. leachii, and D. dentifer over a 10-d-feeding experiment. The strategies for
storage and excretion of Lyngbya compounds by these species have important
implications for understanding the transfer of toxins through marine food
chains.

METHODS AND MATERIALS

Study Sites. Two areas were chosen because they had persistent blooms
of L. majuscula: Deception Bay (DB), on the Western shore of Moreton
Bay (27-050 S, 153-090 E); and Adams Beach (AB), a sheltered area on the
Eastern side of Moreton Bay (27-510 S, 153-410 E). Both sites have extensive sea
grass beds with macroalgae locally dominating in places (Abal et al., 2001).
L. majuscula was collected from these sites and maintained in aquaria at
Moreton Bay Research Station (MBRS).
Study Organisms. S. striatus (Lyngbya exposed) were collected from
L. majuscula blooms at the sites described above in 2002. A second group of
S. striatus was obtained from a prawn pond at Bribie Island Aquaculture
Research Centre (BIARC). Sea hares had been recruited in the pond as larvae
and as there was no apparent L. majuscula growth, they were considered
Bnaı̈ve^ to L. majuscula (Lyngbya naı̈ve). In the laboratory, the Lyngbya-naı̈ve
group of S. striatus was fed a diet of blanched lettuce before our feeding exper-
iments. B. leachii and D. dentifer were collected from a L. majuscula bloom at
Adams Beach. All opisthobranchs were maintained in separate 50-l tanks in a
closed aquarium system at MBRS. Salinities were kept between 34 and 36 ppt
and temperature consistent with ambient (24-C) with 12-hr light and dark
cycles.
Sample Preparation and Chemical Analyses. To quantify L. majuscula
secondary metabolites in mesograzers, animals were starved for 24 hr to allow
evacuation of gut contents before being euthanized in iced salt water. Shells
1598 CAPPER ET AL.

were removed from D. dentifer and discarded. Organisms were dissected into
head and foot (HF) to represent external body parts, and digestive gland (DG) to
represent total viscera (Avila, 1995). These samples were exhaustively extracted
(five times) using 1:1 dichloromethane:methanol. Extracts were rotary evapo-
rated and dried under pure nitrogen before being resolubilized in 1:1
methanol:distilled water and then analyzed by high-performance liquid
chromatographyYtandem mass spectrometry (HPLCYMS/MS).
To determine if L. majuscula secondary metabolites were secreted or
egested by S. striatus and B. leachii, samples of ink, feces, and eggs (where avail-
able) were analyzed. Ink was collected on absorbent filter paper after gently
squeezing an emersed animal between the fingers. Ink was extracted three times
in 1:1 dichloromethane:methanol (Pennings and Paul, 1993a). Replicates were
pooled and dried by rotary evaporation. Fecal samples were collected, frozen,
freeze-dried, and then extracted in acetone three times. Acetone was used, as it
extracts organics from plant-derived matrices due to its hydrophilic properties.
Replicates were pooled and dried by rotary evaporation. The extract was
resolubilized in 1:1 methanol:distilled water and analyzed by HPLCYMS/MS.
To quantify secondary metabolites in L. majuscula, voucher samples from
all assays were frozen at j20-C and then freeze-dried. Plant material was
extracted three times in acetone. Three replicate extracts were dried by rotary
evaporation. Concentrations of secondary metabolites (in milligrams per
kilogram) were determined by HPLCYMS/MS using a PE/Sciex API 300 mass
spectrometer equipped with a high-flow electrospray interface (TurboIonspray)
coupled to a Perkin Elmer series 200 HPLC system. Separation was achieved
using a 150
4.6-mm Altima C18 column (Alltech) run at 35-C, with a mobile
phase consisting of 80/20 acetonitrile/hi-pure water containing 0.1% formic
acid and 2 mM ammonium formate at a flow rate of 0.8 ml minj1. The flow
was split post column such that the flow to the mass spectrometer interface was
250 ml minj1. Under these conditions, the retention times were 11.7 and 9.3 min
for lyngbyatoxin-a and debromoaplysiatoxin, respectively. The mass spec-
trometer was operated in the positive ion, multiple-ion monitoring mode. Ions
monitored with dwell times of 300 msec were 438.3 (M+H)+ and 410.3 for
lyngbyatoxin-a and 543.3 for debromoaplysiatoxin. Quantification was achieved
by comparison to standards of DAT (kindly provided by Dr. R.E. Moore,
Department of Chemistry, University of Hawaii at Manoa) and LTA
(Calbiochem) run under the same conditions. Using a 20-ml injection, the detec-
tion limit for both toxins was typically 0.01 mg kgj1 in the extracted solution.
Partitioning of Secondary Metabolites. Lyngbya-exposed S. striatus (1.31
gwwt T 0.10 SE, N = 9) were housed individually in 1-l glass jars filled with
unfiltered aerated seawater and allowed to acclimate for 48 hr. Seawater was
changed every 2 d. Each sea hare was offered L. majuscula from the Deception
Bay bloom and left to feed for a 10-d-period (Table 1). Voucher samples of
 TABLE 1. SECONDARY METABOLITE CONCENTRATIONS DETECTED IN L. Majuscula USED IN FEEDING ASSAYS

Lyngyatoxin-a Debromoaplysiatoxin
THE FATE OF

Food type and Food weight Day of concentrationb,c concentrationb,c

Assay sourcea ( blotted gwwt)b test (mg kgj1) (mg kgj1)

Internal partitioning assays

S. striatus L. majuscula (DB) 10.0 (T0.01) 1 n/d 0.410 (T0.022)
(Lyngbya exposed) 10 n/d 0.048 (T0.020)
S. striatus Lettuce 9.9 (T0.01) 1 n/d n/d
(Lyngbya naive) 10 n/d n/d
B. leachii L. majuscula (AB) 2.0 (T0.01) 1 0.362 (T0.04) n/d
3.0 (T0.02) 3 7.981 (T0.16) n/d
3.0 (T0.01) 5 8.003 (T2.13) n/d
3.0 (T0.01) 7 1.619 (T0.15) n/d
5.0 (T0.02) 9 1.296 (T0.45) n/d
D. dentifer L. majuscula (AB) 2.0 (T0.01) 1 11.77 (T0.66) n/d
10 40.66 (T5.43) n/d
Whole animal, body secretions and egested material assays
S. striatus L. majuscula (AB) 2.0 (T0.02) 1 1.215 (T0.11) n/d
2.0 (T0.02) 3 0.053 (T0.01) n/d
5.3 (T0.02) 5 n/d n/d
5.0 (T0.03) 7 0.258 (T0.02) n/d
9.9 (T0.02) 9 0.013 (T0.00) n/d
B. leachii L. majuscula (AB) 5.1 (T0.11) 1 4.561 (T0.18) n/d
Lyngbya majuscula TOXINS IN THREE POTENTIAL CONSUMERS

10 3.493 (T0.26) n/d

a
Letters in parentheses refer to location of L. majuscula collected for use in feeding assays: AB, Adams Beach; DB, Deception Bay.
b
Numbers in parentheses refer to standard error values.
c
n/d = not detected by HPLCYMS/MS.
1599
1600 CAPPER ET AL.

L. majuscula were retained on d 1 and d 10 to quantify secondary metabolite

concentrations (Table 1). The second group of S. striatus (0.79 gwwt T 0.06 SE,
N = 9), Lyngbya naı̈ve, was only fed blanched lettuce (Table 1).
B. leachii (0.26 gwwt T 0.03 SE, N = 9) and D. dentifer (0.23 gwwt T 0.03
SE, N = 9) were maintained as described above. B. leachii were fed with fresh
L. majuscula collected from blooms at Adams Beach every 2 d (Table 1), while
D. dentifer received fresh L. majuscula only on d 1 since they only consumed a
small amount during our study. Voucher samples of L. majuscula from each
food change were retained for secondary metabolite quantification (Table 1).
S. striatus (0.11 gwwt T 0.01 SE, N = 9) and B. leachii (1.05 gwwt T 0.09 SE,
N = 9) were collected from the Adams Beach bloom of L. majuscula. S. striatus
were maintained as described above and fed with fresh L. majuscula collected
from Adams Beach every 2 d since they rapidly consumed their food. B. leachii
individuals consumed less L. majuscula and so they were only fed on d 1
(Table 1). Body secretions including ink, eggs (where available), and fecal
matter were collected from each animal at the end of our experiment. Nine
whole animals were pooled to form three replicates for HPLCYMS/MS analy-
sis. Secretions were processed in the same manner.
The mean concentration of secondary metabolites in whole animal,
secretions, and egested material were analyzed using single-factor analysis of
variance (ANOVA), followed by Fisher’s least significant difference (LSD)
pairwise comparison tests. Data sets were subjected to Cochran and Levene’s
homogeneity of variance tests. Data that deviated from a normal distribution
were transformed using ¾mean concentration.

RESULTS

Internal Partitioning of Secondary Metabolites. Significant differences

were observed between the sequestration of secondary metabolites in the
digestive gland ( DG), and head and foot (HF) of S. striatus fed L. majuscula
from Deception Bay (Figure 1A, ANOVA, df1, F318.36, P < 0.001). Both
secondary metabolites (lyngbyatoxin-a and debromoaplysiatoxin) were seques-
tered at equivalent rates (ANOVA, df1, F1.95, P = 0.20). No secondary metabo-
lites were detected in S. striatus fed blanched lettuce. Significant differences
were also observed in the concentrations of lyngbyatoxin-a sequestered between
the DG and HF of B. leachii (Figure 1B, ANOVA, df1, F10.77, P = 0.030).
Diniatys denifer exhibited high individual variability in concentrations of
secondary metabolites sequestered in both DG and HF (Figure 1C) with
lyngbyatoxin-a detected in the DG but not the HF, and debromoaplysiatoxin in
the HF and not in the DG.
THE FATE OF Lyngbya majuscula TOXINS IN THREE POTENTIAL CONSUMERS 1601

FIG. 1. Concentrations of lyngyatoxin-a and debromoaplysiatoxin in digestive gland and

body tissue of (A) S. striatus previously exposed to L. majuscula fed L. majuscula from
Deception Bay and S. striatus not knowingly exposed to L. majuscula fed blanched
lettuce. (B) B. leachii fed L. majuscula from Adams Beach; (C) D. dentifer fed
L. majuscula from Adams Beach. Bars are mean concentration of secondary metabolite
(+SE). Body parts are significantly different from each other in (A) and (B) (single-factor
ANOVA), but not (C). Scales differ between vertical axes.
1602 CAPPER ET AL.

Partitioning of Secondary Metabolites between Whole Animal and

Secretions. Significant differences were observed between whole animal,
secretions, and egested material for both S. striatus and B. leachii (ANOVA,
df1, F441.89, P < 0.001) (Figure 2A,B). Lyngbyatoxin-a concentrations in
S. striatus whole animal (3.94 mg/kgj1) was much greater than secretions
or egested material (ink 0.12 mg/kgj1, fecal matter 0.56 mg/kgj1, and eggs
0.05 mg/kgj1) (ANOVA, df3, F188.93, P < 0.001, Figure 2A). B. leachii

FIG. 2. Concentrations of lyngyatoxin-a (LTA) in whole animal, secretions, and egested

material of (A) S. striatus and (B) B. leachii fed L. majuscula from Adams Beach. Bars
are mean concentrations of LTA (+SE). Significant differences between whole animal
and their bodily secretions are shown (single factor ANOVA). Different letters above the
bars indicate significant differences between tissues, secretions and egested material (P <
0.05) using Fisher’s LSD pairwise comparison tests. Bars with the same letter are not
significantly different. (Eggs were not available from B. leachii during the trial period).
Scales differ between vertical axes.
THE FATE OF Lyngbya majuscula TOXINS IN THREE POTENTIAL CONSUMERS 1603

had lower concentrations of lyngbyatoxin-a (ANOVA, df2, F57.62, P < 0.001,

Figure 2B) in whole animal (2.24 mg/kgj1) compared to that of its ink
(5.41 mg/kgj1) and fecal matter (6.71 mg/kgj1) (Figure 2B).

DISCUSSION

Our results show that S. striatus can sequester and compartmentalize both
lyngbyatoxin-a and debromoaplysiatoxin. The different concentrations of
lyngbyatoxin-a and debromoaplysiatoxin in the digestive gland were lower
than those detected in whole L. majuscula, which may indicate bioaccumulation
from feeding before our experiments. This highlights the importance of diet
history when examining secondary metabolite dynamics in sea hares. Pennings
and Paul (1993a) found that over a 3 wk period the concentration of mal-
yngamides in S. longicauda did not change.
B. leachii has a different strategy for sequestering L. majuscula secondary
metabolites. The digestive gland, ink, and the fecal matter had high concen-
trations of lyngbyatoxin-a. Transfer of lyngbyatoxin-a from body tissue to body
secretions is one potential mechanism for toxin excretion. S. striatus seems to
transform lyngbyatoxin-a and debromoaplysiatoxin into less harmful acetates
(Pennings et al., 1996), but B. leachii may lack the digestive and physiological
ability for transformation and, therefore, excretes these compounds. Lyngbya-
toxin-a in the ink could also provide B. leachii with a defense mechanism
against predation (Johnson and Willows, 1999).
The question of whether opisthobranch mollusks use sequestered sec-
ondary metabolites for defensive purposes has sparked some debate (Paul and
Pennings, 1991; Pennings et al., 1999, 2001; Rogers et al., 2000). Compart-
mentalization of compounds in the digestive gland offers little protection if the
sea hare must be eaten before the predator encounters the deterrent (Pennings
et al., 2001). Pennings et al. (1999) argue that accumulation of extremely high
concentrations of dietary-derived metabolites in the digestive gland are more
likely to assist detoxification of a chemically rich diet than deter predators,
whereas deployment of secondary metabolites to the skin or ink may afford
some defensive protection (Pennings and Paul, 1993a; Rogers et al., 2000).
Ink containing high concentrations of lyngbyatoxin-a excreted by B. leachii
might provide a defensive role; however, we suggest that this transfer might be
an excretory mechanism employed by B. leachii to avoid toxicity. B. leachii
demonstrated increasing growth rates over a 10-d period in the laboratory when
fed a monospecific diet of L. majuscula. This reached a plateau between 10 and
20 d (Capper et al., unpublished data) with the death of the animal following
soon after (personal observation). While morbidity may be related to the
1604 CAPPER ET AL.

toxicity of L. majuscula, it could also be related to incomplete nutritional

requirements. Dense aggregations of B. leachii are found in local L. majuscula
blooms. This may reflect exploitation of an abundant food source rather than
dietary specialization. It is more likely that B. leachii consumes a mixed diet in
the field (Paige, 1988), possibly getting more nutrients and preventing harmful
additive accumulation of secondary metabolites in the digestive gland. The
consequences of debromoaplysiatoxin consumption in B. leachii are unknown,
as none was detected in L. majuscula used in these feeding trials.
Lyngbyatoxin-a and debromoaplysiatoxin found in D. dentifer after it ate
L. majuscula for 10 d showed high individual variability. This may be related to
the palatability of L. majuscula used in our feeding trials. However, a lack of
uniformity in the distribution of secondary metabolites among test animals may
suggest D. dentifer are not preferential consumers of L. majuscula. D. dentifer
may use L. majuscula as a source of refuge from predation rather than a food
source (Cruz-Rivera and Paul, 2002). Little is known of the dietary repertoire in
this species and fate of L. majuscula secondary metabolites.
Debromoaplysiatoxin was the predominant toxin in L. majuscula blooms at
Deception Bay during our experiments, but lyngbyatoxin-a was also detected.
Lyngbyatoxin-a and debromoaplysiatoxin had previously been detected at
Adams Beach, however, only lyngbyatoxin-a was detected during these
experiments. This variation in L. majuscula secondary metabolites highlights
their temporal and spatial variability.
Our results show distinct differences in sequestration mechanisms utilized
by three species of opisthobranchs fed L. majuscula. This suggests that the
generic sequestration hypothesis may not be applicable to all opisthobranch
mollusks. Implications for bioaccumulation or biomagnification of lyngbya-
toxin-a and debromoaplysiatoxin through the marine food web warrant further
investigation in both B. leachii and S. striatus, particularly following mass
mortalities after the disappearance of L. majuscula blooms. High quantities of
lyngbyatoxin-a egested via fecal matter in B. leachii may also result in the
introduction of L. majuscula secondary metabolites to phagotrophic or detrital
food webs.

AcknowledgmentsVThis project has been supported by Centre for Marine Studies Morteon
Bay Research Station Scholarship; South East Queensland Regional Water Quality Management
Strategy (SEQRWQMS), Brisbane, Australia; and Graduate Research Student Travel Award,
University of Queensland. The authors thank the following people for their help: Dr. Wayne Knibb
and Daniel Willett for supply of Lyngbya-naı̈ve S. striatus from Bribie Island Aquaculture Research
Centre (BIARC); Dr. Clay Carlson for assistance in identification of D. dentifer; Mr. Geoff
Eaglesham and Mr. Nick Osborne for help with toxicology work at National Research Centre for
Environmental Toxicology (EnTox); Dr. Nazim Khan (University of Queensland) for help and
guidance with statistics; Prof. Valerie Paul and Mr. Raphael Ritson-Williams for feedback and
critique of the manuscript.
THE FATE OF Lyngbya majuscula TOXINS IN THREE POTENTIAL CONSUMERS 1605

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