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Interaction of chemical and structural components

providing defences to sea pansies Renilla reniformis
and Renilla muelleri
a a b
Etiene E.G. Clavico , Bernardo A.P. Da Gama , Angélica R. Soares , Keila Mara
c a
Cassiano & Renato C. Pereira
a
Departamento de Biologia Marinha, Universidade Federal Fluminense, Niterói, Rio de
Janeiro, Brazil
b
Núcleo em Ecologia e Desenvolvimento Sócio Ambiental de Macaé (NUPEM),
Universidade Federal do Rio de Janeiro, Macaé, Rio de Janeiro, Brazil
c
Departamento de Estatística, Instituto de Matemática, Universidade Federal
Fluminense, Niterói, Rio de Janeiro, Brazil

To cite this article: Etiene E.G. Clavico , Bernardo A.P. Da Gama , Angélica R. Soares , Keila Mara Cassiano & Renato C.
Pereira (2013): Interaction of chemical and structural components providing defences to sea pansies Renilla reniformis
and Renilla muelleri , Marine Biology Research, 9:3, 268-275

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 Marine Biology Research, 2013; 9: 268
275

ORIGINAL ARTICLE

Interaction of chemical and structural components providing defences

to sea pansies Renilla reniformis and Renilla muelleri

ETIENE E.G. CLAVICO1, BERNARDO A.P. DA GAMA1, ANGÉLICA R. SOARES2,

KEILA MARA CASSIANO3 & RENATO C. PEREIRA1*
1
Departamento de Biologia Marinha, Universidade Federal Fluminense, Niterói, Rio de Janeiro, Brazil, 2Núcleo em Ecologia
e Desenvolvimento Sócio Ambiental de Macaé (NUPEM), Universidade Federal do Rio de Janeiro, Macaé, Rio de Janeiro,
Brazil, and 3Departamento de Estatı́stica, Instituto de Matemática, Universidade Federal Fluminense, Niterói, Rio de
Janeiro, Brazil
Downloaded by [Etiene Clavico] at 14:21 06 December 2012

Abstract
Chemical and structural defence mechanisms are reported to co-occur in both terrestrial and marine systems. Among
benthic marine organisms, the common co-occurrence of secondary metabolites and calcium carbonate (CaCO3) in soft
corals provides an opportunity for testing synergistic interactions between these traits. Defensive properties of crude
extracts, chemical fractions and CaCO3 sclerites from two sympatric species of soft corals, Renilla reniformis and R.
muelleri, from Guanabara Bay, southeastern Brazil, were examined against fishes. To evaluate a potential interaction
(secondary metabolites versus sclerites), both crude extracts and sclerites were evaluated as isolated defences and in
combination in field assays against generalist fishes during the austral summer of 2007. While neither sclerites nor
crude extracts from R. reniformis deterred feeding when offered individually in artificial food, both traits from R. muelleri
offered individually provided effective defence. For both species, however, the combination of secondary metabolites
and sclerites significantly deterred feeding, indicating that these traits are more effective in combination than in
isolation.

Key words: Chemical defence, physical defence, synergism, sea pansies

Introduction Octocorals produce several types of compounds,

Biological interactions are important aspects regu-
lating marine populations and chemical mediations
are known to be important principally in the main-
tenance of benthic marine communities (Paul et al.
2007; Hay 2009). Predator
prey interactions are
common ecological processes often driven by sec-
ondary metabolites (Paul et al. 2011).
Defensive chemicals are commonly present among
marine invertebrates from low (Freeman & Gleason
2010) to high latitudes (McClintock et al. 2010),
and appear to be most common among vulnerable
species living under high predation, such as sponges
(Leong & Pawlik 2010), sea slugs (Takagi et al.
2010), tunicates (Nunez-Pons et al. 2010) and soft
corals (Changyun et al. 2008).
including sesquiterpenes, diterpenes and acetogen-
ins (Blunt et al. 2011), which seem to play important
roles in protecting their respective colonies against
generalist consumers (e.g. Kelman et al. 1999; Koh
et al. 2000). For example, of 32 species of Caribbean
gorgonians evaluated, all yielded active extracts
against predation (O’Neal & Pawlik 2002).
Structural skeletal components of marine inverte-
brates have also been investigated as potential
defensive mechanisms against predation (Harvell
et al. 1988), but the results are sometimes contro-
versial. For example, purified calcitic sclerites from
the gorgonian Pseudopterogorgia acerosa inhibited
feeding (Harvell et al. 1988), although it has been
found that sclerites from 30 out of 32 Caribbean
gorgonian species (including P. acerosa) were pala-

*Correspondence: Renato C. Pereira, Departamento de Biologia Marinha, Universidade Federal Fluminense, PO Box 100.644, 24001-
970, Niterói, Rio de Janeiro, Brazil. E-mail: renato.pereira@pq.cnpq.br or etieneclavico@gmail.com
Published in collaboration with the University of Bergen and the Institute of Marine Research, Norway, and the Marine Biological Laboratory,
University of Copenhagen, Denmark

(Accepted 10 September 2012; Published online 3 December 2012; Printed 10 December 2012)
ISSN 1745-1000 print/ISSN 1745-1019 online # 2013 Taylor & Francis
http://dx.doi.org/10.1080/17451000.2012.739693

table to fish (O’Neal & Pawlik 2002). These contra-
dictory results have been attributed to factors such as
methodological differences and chemical aspects
(O’Neal & Pawlik 2002). Sclerites contain CaCO3
that may act as a chemical deterrent (Hay et al.
1994; Schupp & Paul 1994), because it would act to
neutralize the low pH of some fish guts and the large
amount of carbon dioxide released in the process
could act as a chemical feeding deterrent. Thus, the
term mineral defence would help to distinguish the
chemical effect from that of CaCO3 acting as a
hardening agent (Schupp & Paul 1994).
Chemical and structural defences have been re-
ported to co-occur in several benthic marine inverte-
brates (e.g. Harvell et al. 1988; Harvell & Fenical
1989; Van Alstyne & Paul 1992; Van Alstyne et al.
1994; Koh et al. 2000; Puglisi et al. 2000, 2002).
Depending on the effect, the interactions between
these agents can be additive, synergistic or antag-
Downloaded by [Etiene Clavico] at 14:21 06 December 2012
onistic. Interactions are additive if the joint action of
the agents does not differ from the sum effect of each
agent in isolation (Berenbaum 1988), and synergistic
and antagonistic interactions occur when the joint
action of different substances is significantly greater
or less than their sum effect (Wilkinson 1973).
Potential synergisms between CaCO3 structures
and chemical defences could be widespread given the
frequent co-occurrence of these two characteristics
among benthic marine organisms. Despite this, sy-
nergistic phenomena have received limited attention
by chemical ecologists. Evidence for synergism be-
tween chemical defences and CaCO3 in feeding
studies has been reported for the green seaweeds
Rhipocephalus phoenix and Udotea cyathiformis (Hay et
al. 1994), the gorgonian Leptogorgia virgulata (Ger-
hart et al. 1988), and has also been found in some
species of sponges (Hill et al. 2005; Jones et al. 2005).
Pennatulacean octocorals belonging to the genus
Renilla are very conspicuous members of sublittoral
soft-bottom communities (Morin et al. 1985). Spe-
cies of Renilla produce secondary metabolites (Keifer
et al. 1986; Barsby & Kubanek 2005) and also
possess structural components, providing a valuable
opportunity to evaluate the action of these compo-
nents as defensive strategies against consumers.
Species of Renilla, such as R. muelleri Koliker, 1872
and R. reniformis (Pallas, 1766), are more character-
istic members of the tropical subtidal fauna of
shorelines exposed to ocean swell (Kastendiek
1976). At Guanabara Bay, Brazil, sympatric R.
reniformis and R. muelleri are two of the dominant
sessile macroinvertebrates present (Da Gama et al.
1995). However, ecological research with these
organisms has rarely been performed. Recent studies
show that briarane diterpenoids from the genus
Renilla protect this sea pansy from predation by
Chemical and structural defences in sea pansies 269
fish and crabs in laboratory assays (Barsby &
Kubanek 2005). Ecological research conducted
with R. muelleri from Guanabara Bay revealed
significant phenotypic plasticity on its sclerites that
act as a physical defence against an array of generalist
fishes in field assays (Clavico et al. 2007).
The main goal of this study was to investigate
the potential chemical and physical defences of
R. reniformis and R. muelleri against fishes in field
feeding assays. In addition, we also evaluated the
potential interaction of physical and chemical traits
to provide increased protection from predatory
fishes. We thus hypothesized that the combined
inhibitory effect of both traits on fish predation is
greater than the effect of either trait alone.
Materials and methods
Organisms and collection sites
The soft-bodied, umbrella-shaped sea pansies
Renilla muelleri and R. reniformis are brightly
coloured and often found on mud or sandy mud
substrata. They are well known to exhibit biolu-
minescence. The colonies of R. muelleri and
R. reniformis used in this study were collected at
Guanabara Bay, a prominent coastal bay located in
the southeastern coast of Brazil (22841?23806?S
and 43802?43818?W) in November 2006 by hand
during scuba (as on p. 4) dives.
Extraction of chemicals and isolation of sclerites
After collection, all samples were placed in seawater
and returned to the laboratory where volumes
(determined by water displacement in a graduated
cylinder) and wet masses were recorded before being
frozen for storage (the work was based on volumetric
amounts rather than on the number of colonies).
Before the extraction process, the relationship be-
tween wet weight and volume was set to ensure that
the aliquots of extract or sclerites added to the
artificial food were performed at volumetrically
natural concentrations.
After defrosting, sea pansies were repeatedly ex-
tracted (successive extraction, 3 times, during 7 days),
with pure acetone. A multiple of 20 ml by volume was
used for extraction, which is the volume of the
Plexiglas mould for artificial food pellets, according
to the relationship previously set between weight and
volume. Multiple Renilla colonies were extracted in
order to prepare each food pellet set. Although this is
the standard procedure used in the vast majority of the
studies in marine chemical ecology, the ideal form
would be that each colony generated one pellet.
However, the colonies from both Renilla species are
 270 E.E.G. Clavico et al.
too small to allow individual extraction. The only
practical consequence of this is that possibly variable
individual concentrations of compounds or sclerites
were averaged, perhaps generating less variability on
the stimulus side, but this would not necessarily elicit a
different response in the measured variable, i.e. fish
consumption between controls and treatments (see
Feeding assays).
The extracts were filtered and solvents eliminated in
a rotary evaporator under reduced pressure. Bioassay-
guided fractionation was performed through a
liquid
liquid partition with methanol (MeOH):hex-
ane 1:1 and again with aqueous MeOH:dichloro-
methane (DCM) 1:1 (MeOH:H2O:DCM 0.5:0.5:1).
Although the study of Renilla feeding deterrent
compounds by Barsby & Kubanek (2005) used a 2:1
mix of MeOH:DCM, we opted to first use acetone, a
rather polar solvent, and later refine the extraction
with wider polarity mixtures. Even though the first
Downloaded by [Etiene Clavico] at 14:21 06 December 2012
extraction was performed with acetone only, possibly
missing very non-polar (or even very polar) com-
pounds, acetone has been successfully used to extract
active compounds from octocorals (e.g. D’Armas et
al. 2008), and activity was indeed recorded with
acetone extracts (see Results).
The sclerite content was obtained by placing the
colonies in 50-ml tubes containing 0.25 N NaOH,
following the work of Alonso (1979). This solution
was then heated to the boiling point. The sclerites
from each sample were rinsed at least five times with
distilled water to remove any traces of NaOH, dried
by heating to 1008C until a steady dry weight was
obtained.
Feeding assays
A total of 8 field experiments were performed,
namely: for Renilla muelleri: (1) chemistry versus
control, (2) sclerites versus control, (3) chemistry
plus sclerites versus control, (4) MeOH fraction
versus control, (5) DCM fraction versus control for
R. reniformis: (6) chemistry versus control, (7)
sclerites versus control, (8) chemistry plus sclerites
versus control. No experiments with fractions from
R. reniformis were performed because experiment 6
has not shown any chemical deterrence. For each
experiment, a set of 20 replicated experimental units
(EU) was prepared, each consisting of double-baited
ropes as further described below.
Previous studies addressing chemical and struc-
tural defences in octocorals have offered test fishes a
choice between one treatment (chemical and/or
structural components) and a control in paired
assays (e.g. Pawlik et al. 1987; Harvell et al. 1988;
Fenical & Pawlik 1991; Pawlik & Fenical 1992; Koh
et al. 2000; Puglisi et al. 2000; Epifanio et al., 1999,
2000). Our field experiments were conducted in a
similar fashion at Cabo Frio Island, Arraial do Cabo
(Rio de Janeiro state, 22859?S42800?W), in the
austral summer of 2007, at 3
6 m depth. Although
Renilla colonies were collected from Guanabara Bay,
this organism also occurs at Arraial do Cabo.
Artificial food pellet preparation was performed
according to Pawlik et al. (1995) and modified
according to Clavico et al. (2006, 2007), i.e. basically
tuna fish substituted squid mantle. In order to
reproduce the same concentrations of chemicals
and sclerites as found in the sea pansies, volumetric
calculations were employed. The sample volume was
determined by water displacement in a graduated
cylinder, i.e. the colonies were added to a graduated
cylinder filled with distilled water until the desired
volume was obtained. This volume was then ex-
tracted to obtain sclerites and extracts (see Extraction
of chemicals and isolation of sclerites). The amount
of extract or sclerite material was thus added to the
artificial food at volumetrically appropriated levels.
The artificial food pellets for all field experiments
were prepared according to the following general
protocol: a mixture composed of 0.625 g of carra-
geenan powder, 5 ml of tuna fish and 15 ml of
distilled water yielded a final volume of about 20 ml.
This mixture was heated to the boiling point, stirred
and allowed to cool down to about 408C (in order to
avoid thermal degradation of compounds) prior to
the addition of (i) extract, (ii) fraction, (iii) sclerite, or
(iv) extract plus sclerite aliquots. The mixture was
then vigorously stirred again and poured into a
rectangular Plexiglas mould (5.05.0 0.5 cm,
internal dimensions) for pellet preparation (2.5 
1.0 0.5 cm). As each mould makes 10 pellets, this
procedure was performed twice to yield a total of 20
replicates for each of the 8 field experiments; there-
fore each set of 20 replicates contained either extract,
sclerites, extract and sclerites, or fraction obtained
from 20 ml of Renilla colonies. Each pellet had a fine
natural fibre line inside for later attachment to EU
nylon ropes. Control pellets, to be paired in each
nylon rope, were made in the same way but without
the addition of crude extract, fraction or sclerites. In
order to ensure uniformity of factors related to
predation in field assays, the same amount of solvent
used to dissolve the extract or fraction into treatment
foods was added to their respective control foods.
This is usually necessary to ensure that no residual
effect of solvents could possibly be confused with the
experimental treatments.
Each EU consisted of a nylon rope with a lead
weight (50 g) at one end and a pair of artificial food
pellets (a control and a treatment) at the other
(upper) end. These pellets were always one control
and one of three types of treatment: (a) secondary

metabolites (crude extract or fraction; CE or F), (b)
sclerites (S), and (c) a combination of sclerites plus
crude extracts (CES). The pairs (controltreat-
ment) were then tied to the nylon ropes (N20
replicates per experiment) and set on the seabed at
least 2 m apart from each other early in the morning,
so as to be exposed to generalist consumers (fish)
long enough to observe a measurable consumption
(usually about 3 h). For this purpose, experiments
were continuously monitored by divers from a
distance great enough to avoid deterring fish from
approaching the pellets, but close enough to allow
observation (about 5 m for average underwater
conditions). After this period all EUs were removed
and the remaining pellets measured with a caliper
and converted to percent of mass eaten for graphical
purposes only. Despite the use of 20 replicates at the
beginning of each field experiment, the final number
of replicates retrieved ranged from 9 to 20 depending
Downloaded by [Etiene Clavico] at 14:21 06 December 2012
on the experiment, as indicated in each graph (see
Results). This was usually due to changing weather
conditions during experiments, which changed un-
derwater conditions and caused poor visibility.
The main generalist fishes at Arraial do Cabo
which might have been able to consume the pellets
are Abudefduf saxatilis (Linnaeus, 1758), Haemulon
aurolineatum (Cuvier 1829), H. steindacneri (Jordan
& Gilbert, 1882), Halichoeres poeyi (Steindachner,
1867) and Chaetodon striatus (Linnaeus, 1758)
(Floeter et al. 2003). The food pellets were watch-
ed by scuba divers, and the natural assemblage
of fish consumers was closely observed. As reef
fishes (Abudefduf saxatilis, Haemulon aurolineatum,
H. steindacneri, Halichoeres poeyi, Chaetodon striatus)
usually exhibit territorial behaviour (e.g. Low 1971)
the minimum distance between food pellets (2 m)
was set taking this into account, so that consumption
of one food pellet was independent of the consump-
tion of the others.
Chemical analyses of anti-feeding compounds
All the crude extracts and fractions obtained were
investigated by Thin Layer Chromatography (TLC)
(Merck, silica gel GF254) and by 1H NMR (300
MHz) spectroscopy (Pavia et al. 2001; Shachar-
Hillm 2001).
Statistical analysis
As in each field experiment the data were obtained in
a paired form (i.e. each treatment food had a control
on the same nylon rope), the results from the field
experiments were analysed by the Wilcoxon signed
ranks test, a nonparametric equivalent to the t-test
for dependent samples, as variables generally did not
Chemical and structural defences in sea pansies 271
follow the assumption of normal distribution (Zar
1999). However, t-tests were also performed (with
transformed data whenever needed) to double-check
results, always leading to the same inference (data
not shown). Differences among treatments were
considered to be significant only when p B0.05
(a5%). For empirical comparison of effectiveness
between individual traits (chemical defences, struc-
tural defences) and combined traits, effect sizes (ES)
were calculated for each experimental result accord-
ing to post-hoc power analysis for t-tests for depen-
dent samples (Faul et al. 2007; Gerrodette 2011).
Statistica 7 and G*Power 3 softwares were employed
for statistical tests and ES calculations, respectively.
Results
Chemical analyses
The determination of the chemical profiles of the
crude extracts from Renilla muelleri and R. reniformis
by 1H NMR revealed the presence of sterols and
glycosyl compounds as major components in both
extracts. Terpenes were also observed, although
as minor components. The 1H NMR spectrum of
the active MeOH fraction obtained from the first
partition of R. muelleri crude extract showed
characteristic signals of glycosyl compounds as
major components. The deterrent DCM fraction,
obtained from the second liquid
liquid partitioning
(MeOH:H2O:DCM 0.5:0.5:1) detected the pre-
sence of terpenes as major components. NMR
spectra of crude extracts and fractions from both
Renilla species are available online as Supplementary
Material.
Field feeding assays
plus chemicals

chemicals, sclerites, and sclerites
Both crude extract and sclerites from Renilla muelleri
were shown to deter fish predation in field assays
(Wilcoxon test, p 0.03 and p0.003, respectively;
Figure 1). In addition, evidence of an interaction
between sclerites and crude extract enhancing de-
fence against fish predation were found for R.
muelleri (Wilcoxon test, p B0.001; Figure 1).
The liquid
liquid partition (MeOH:H2O:DCM
0.5:0.5:1) performed using R. muelleri crude extracts
revealed deterrent properties against fish from the
DCM fraction (Wilcoxon test, p B0.05; Figure 2).
Both assays conducted with crude extract alone
and sclerites alone from Renilla reniformis did not
deter consumption by generalist fishes (Wilcoxon,
p 0.05; Figure 3). Therefore, no chemical fractio-
nation was conducted with the extract from this
species. However, when sclerites and crude extract
 272 E.E.G. Clavico et al.
90
Treatment 90
Treatment N=9
80 Control
80 Control N.S.
70 N = 12
70
60 p = 0.03 N = 19 N = 18
60 N = 10
p < 0.001 p < 0.001
% eaten

50 N.S.

% eaten
N = 17 50
40 p = 0.003 40
30 30
20 20
10 10
0 0
Chemistry Sclerites Chemistry+Sclerites Chemistry Sclerites Chemistry+Sclerites
Renilla muelleri Renilla reniformis

Figure 1. Treatment effects (chemistry, sclerites and chemistry
plus sclerites) from Renilla muelleri on feeding by generalist fishes
in field assays. j Treatment and h Control. Data are expressed as
mean% eaten plus standard error (SE). N number of replicates
retrieved per experiment, and p-values are from Wilcoxon signed
ranks tests.
Downloaded by [Etiene Clavico] at 14:21 06 December 2012
Figure 3. Treatment effects (chemistry, sclerites and chemistry
plus sclerites) from Renilla reniformis on feeding by generalist
fishes in field assays. j Treatment and h Control. Data are
expressed as mean% eaten plus standard error (SE). N number
of replicates retrieved per experiment, and p-values are from
Wilcoxon signed ranks tests. NS, non-significant result.

from R. reniformis were combined, there was a
significant inhibition of consumption (Wilcoxon
test, pB0.01; Figure 3). The combined effect sizes
of chemistry and sclerites were higher than effect
sizes of either trait isolated for R. muelleri (Figure 4).
For R. reniformis, there was also a strong effect size of
combined traits (Figure 5), while isolated traits did
not deter fish feeding.
Discussion
The genus Renilla comprises distinct and ancient
pennatulacean octocorals. Despite their conspicuous
presence in sublittoral soft-bottom environments,
these animals have rarely been studied from an
ecological standpoint (see Kastendiek 1976, 1982;
Creed & Coull 1984; Keifer et al. 1986; Barsby &
Kubanek 2005; Clavico et al. 2007 for exceptions),
although there are over 700 papers reporting studies
on, or applications of Renilla bioluminescence. Our
research results with sympatric Renilla reniformis and
R. muelleri from Guanabara Bay (Brazil) suggests
that both species were protected from fish predators
through a combination of structural defences

CaCO3 sclerites
and chemical defences. Both traits
appear to play a joint role in Renilla protection.
Data from the feeding experiments, using the
natural crude extract and sclerite concentrations
found in living colonies, revealed that crude extracts
or sclerites alone provided significant protection to
DCM *

90 MeOH
Treatment
80 Control
R. muelleri

70 N = 20 C+S *
N.S.
60
N = 17
% eaten

50 p < 0.05
Sclerites *
40
30 Chemistry *
20
-0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0
10
Effect size
0
MeOH DCM Figure 4. Estimated effect sizes (ES) of distinct Renilla muellerii
Renilla muelleri defensive traits. Positive ES indicates that the trait inhibited
consumption by fishes in field assays, while negative ES indicated
Figure 2. Treatment effects from semi-purified metabolites that such a trait actually stimulated consumption. * indicates
(MeOH and DCM fractions) from Renilla muelleri on feeding by significant effects (p B0.05) according to Wilcoxon signed ranks
generalist fishes in field assays. j Treatment and h Control. Data tests comparing treatment and control consumption. C S 
are expressed as mean% eaten plus standard error (SE). N treatment containing crude extracts (Chemistry) and Sclerites.
number of replicates retrieved per experiment, and p-values are MeOH and DCM refer to methanol and dichloromethane
from Wilcoxon signed ranks tests. NS, non-significant result. fractions, respectively.

C+S
R. reniformis
Sclerites
Chemistry
-0.4 -0.2 0.0 0.2 0.4 0.6
Effect size
Figure 5. Estimated effect sizes (ES) of Renilla reniformis defen-
sive traits. Positive ES indicates that the trait inhibited consump-
tion by fishes in field assays, while negative ES indicated that such
a trait actually stimulated consumption. * indicates significant
effects (p B0.05) according to Wilcoxon signed ranks tests
comparing treatment and control consumption. C S treat-
ment containing crude extracts (Chemistry) and Sclerites.
Downloaded by [Etiene Clavico] at 14:21 06 December 2012
R. muelleri against predation by generalist fishes. In
addition, the defensive properties of sclerites and
crude extracts when tested together were slightly
higher than when evaluated individually. In contrast,
it has been shown that neither chemical nor physical
structures alone provided defence against fish
predation to the congeneric sea pansy R. reniformis,
although they significantly deterred fish feeding
when tested together.
Chemical and physical defences of marine sessile
organisms have long been a subject of ecological
research (e.g. Bridwell 1918; Janzen 1969; Pennings
& Paul 1992). Although the use of secondary
metabolites by octocorals as defensive agents against
predation has been well established in the literature
(Harvell & Fenical 1989; Pawlik 1993; Bolser & Hay
1996; Koh et al. 2000; Barsby & Kubanek 2005), the
role of calcite sclerites of octocorals as defensive
agents against potential predators remains unclear
(see Sammarco et al. 1987; Harvell et al. 1988;
Harvell & Fenical 1989).
Beyond being utilized as a defensive strategy, other
functions are often attributed to alcyonacean scler-
ites. For example, the primary function of sclerites is
structural support for Sinularia colonies, leaving the
defensive function to the secondary metabolites (Van
Alstyne et al. 1994). Our results indicate that in
isolation neither secondary metabolites nor sclerites
of R. reniformis are able to provide defence against
predation.
Both large and densely packed sclerites have been
suggested to turn octocorals less palatable to pre-
dation than shorter, less dense ones (West 1998).
Previous research indicated that sclerites of
R. reniformis are smaller but more densely packed
than sclerites of R. muelleri (Clavico et al. 2007). The
Chemical and structural defences in sea pansies 273
larger, less densely packed sclerites of R. muelleri
* exhibited defensive properties and thus do not
support the idea that different sclerite densities can
provide different degrees of anti-predatory protec-
tion (Clavico et al. 2007), but the reduced sclerites
from R. reniformis could reduce their defensive
properties, despite their high densities. It is therefore
noteworthy that these sclerites only deterred fish
feeding when combined with crude extracts.
The defensive strategy of marine invertebrates of
0.8 1.0 the subclass Octocorallia is frequently linked to
secondary metabolites, especially to terpenoidic
compounds (Sammarco et al. 1987; Harvell et al.
1988; Harvell & Fenical 1989; Sammarco & Coll
1992; Barsby & Kubanek 2005). In fact, terpenes
have been detected as conspicuous compounds in
R. muelleri through 1H NMR analyses. Our work
suggests, in agreement with other studies (e.g.
Barsby & Kubaneck 2005), that they can also be
responsible for the deterrent properties found in
R. muelleri. The bioassay-guided fractionation of the
crude extract from R. muelleri yielded a DCM active
fraction, shown to be rich in terpenoidic com-
pounds. According to 1H NMR analyses, terpenes
have also been detected in the crude extract from
R. reniformis. However, deterrent properties were
not observed when this extract was tested in isola-
tion. The absence of activity from the chemical
defences of R. reniformis could be due to qualitative
and/or quantitative differences of the active com-
pounds, when compared to the extract of R. muelleri.
Interactions between physical and chemical agents
can be additive (Berenbaum 1988) or synergistic
(Gerhart et al. 1988), depending on the defensive
result shown by the interaction of the components.
Clearly, analyses of only one defensive characteristic
(such as sclerites or crude extracts) in isolation can
lead to a misunderstanding about anti-predator
defences in benthic marine invertebrates. Our data
have shown that the combination of chemical
compounds and sclerites from both species of
octocorals, although acting differently, provides
defence against predation by decreasing fish feeding.
In experiments with R. reniformis, when neither
crude extract nor sclerites deterred predation (tested
in isolation), one could conclude that this octocoral
is not defended, although our data showed that this
is not true. The joint defensive property from
R. reniformis was not attributable to the isolated
action of structural components or secondary meta-
bolites, but our data suggest that the combination of
the chemical and structural components of this
species exhibited a synergistic effect. These results
are similar to previous studies, which have shown
that chemical and physical components of octocorals
of the genus Leptogorgia do not deter feeding when
 274 E.E.G. Clavico et al.
offered separately to fishes, but act together to deter
fishes when offered in combination (Gerhart et al.
1988). Our results expand this finding to Renilla and
indicate that, at least for R. reniformis, the sclerites
and crude extract interact synergistically to defend
sea pansies against predation by generalist fishes.
Some previous investigations have shown that
both CaCO3 and chemical characteristics can affect
prey susceptibility to predation (e.g. Pennings &
Paul 1992; Schupp & Paul 1994), but most have not
addressed why these two traits co-occur so com-
monly. A presumptive interpretation for the activity
of small sclerites of R. reniformis, when aggregated
with the crude extract, is that they can abrade the
protective mouth or gut linings or modify gut pH of
potential predators, potentially facilitating the action
of bioactive secondary metabolites, but this needs
further investigation. Moreover, it remains to be
demonstrated whether an additional Renilla trait,
Downloaded by [Etiene Clavico] at 14:21 06 December 2012
bioluminescence, plays a defensive role in this group,
and how this trait interacts with sclerites and
chemical defences.
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