The purple pigment aplysioviolin in sea hare ink deters predatory blue crabs through their chemical senses
ABSTRACT Sea hares release an ink secretion composed of purple ink and white opaline as a potential chemical defence against predators. The aim of our study was to identify deterrent molecules in the ink of Aplysia californica against an allopatric generalist crustacean predator, the blue crab Callinectes sapidus, and to define the mechanisms of action of the deterrents against crabs. We used two behavioural assays, a squirting assay and an ingestion assay, to show that ink is highly effective and that opaline is moderately effective in suppressing feeding of crabs. Results with reversibly blinded crabs demonstrate that the deterrence is mediated through the crabs’ chemical senses. We used bioassay-guided fractionation to identify the purple molecules aplysioviolin and phycoerythrobilin as a major and minor deterrent, respectively, in ink against crabs. These molecules derive from a light-harvesting protein in the photosynthetic system of dietary algae. This is the first demonstration of an animal converting a photosynthetic pigment into a chemical deterrent. Mixing opaline and ink enzymatically produces hydrogen peroxide, which also functions as a chemical deterrent against crabs. Our results and those of other studies show that sea hares use a diversity of molecules in their skin, mucus and ink secretion to chemically defend themselves against their potential predators. Aplysioviolin, phycoerythrobilin and hydrogen peroxide also exist in ink secretion of Aplysia dactylomela, a sea hare sympatric to blue crabs, and thus we posit that these molecules are potentially effective in ecologically relevant predator–prey interactions and need to be scrutinized in more ecologically relevant experiments.
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ABSTRACT: Chemical defenses are used by many organisms to avoid predation, and these defenses may function by stimulating predators' chemosensory systems. Our study examined detection mechanisms for components of defensive ink of sea hares, Aplysia californica, by predatory sea catfish, Ariopsis felis. Behavioral analyses show aplysioviolin and phycoerythrobilin are detected intra-orally and by barbels and are deterrent at concentrations as low as 0.1% full strength. We performed electrophysiological recordings from the facial-trigeminal nerve complex innervating the maxillary barbel and tested aplysioviolin, phycoerythrobilin, amino acids, and bile salts in cross-adaptation experiments. Amino acids and bile salts are known stimulatory compounds for teleost taste systems. Our results show aplysioviolin and phycoerythrobilin are equally stimulatory and completely cross-adapt to each other's responses. Adaptation to aplysioviolin or phycoerythrobilin reduced but did not eliminate responses to amino acids or bile salts. Adaptation to amino acids or bile salts incompletely reduced responses to aplysioviolin or phycoerythrobilin. The fact that cross-adaptations with aplysioviolin and phycoerythrobilin were not completely reciprocal indicates there are amino acid and bile salt sensitive fibers insensitive to aplysioviolin and phycoerythrobilin. These results indicate two gustatory pathways for aplysioviolin and phycoerythrobilin: one independent of amino acids and bile salts and another shared with some amino acids.Journal of Comparative Physiology 12/2011; 198(4):283-94. · 1.86 Impact Factor
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ABSTRACT: Many animals release secretions in defense against predators. Some marine molluscs, including cephalopods (squid, octopus, cuttlefish) and gastropods (sea hares), release a colored ink secretion. Observational evidence supports the idea that inking is a defensive behavior that protects cephalopods from predators by forming a visual smokescreen or visual mimic (pseudomorph). Another possible function of cephalopod ink is to act against the chemical senses of predators either as a deterrent or distracting food mimic (phagomimic). Experimental tests of both hypotheses are lacking for cephalopods. In our study, we tested the hypothesis that squid use ink as a defense against attacks by predatory fish by performing three sets of experiments to examine the behavior of juvenile French grunts, Haemulon flavolineatum, toward ink from Caribbean reef squid, Sepioteuthis sepioidea. In the first set of experiments, a pseudomorph assay, in which ink was presented between a fish and a piece of food, assessed effects of ink on the approach and capture phase of a predator's attack. This showed that a pseudomorph of squid ink hindered the attack by significantly delaying food capture as well as evoking significantly more avoidance of or biting at the pseudomorph compared to a control pseudomorph of carboxymethylcellulose. A pseudomorph of carboxymethylcellulose plus food color to simulate the color of squid ink had a similar effect to the squid ink pseudomorph. In a second set of experiments, a disc assay, in which ink was added to meat-flavored paper discs, examined ink's effect on the consumption of food, simulating ink's protective effect if a squid and its ink are taken into a predator's mouth. This showed that squid ink added to meat-flavored discs significantly changed handling of the discs and increased, though non-significantly, their rejection. The same food color as used in the pseudomorph assay, when added to meat-flavored discs, significantly affected handling and rejection of the discs, showing that the food color itself, intended as a control, is unpalatable. In the third set of experiments, the disc assay was used to show that ink did not increase the acceptance of unflavored (i.e. without meat) discs, a result suggesting that ink is not a phagomimic. Our study presents the first experimental results supporting the hypothesis that inking protects squid against predatory fish, and that it acts during both the capture and consummatory phases of attacks: during the capture phase through visual and/or chemical effects against predators, and during the consummatory phase through unpalatable chemicals.Journal of Experimental Marine Biology and Ecology. 01/2010;
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ABSTRACT: Chemicals are a frequent means whereby organisms defend themselves against predators, competitors, parasites, microbes, and other potentially harmful organisms. Much progress has been made in understanding how a phylogenetic diversity of organisms living in a variety of environments uses chemical defenses. Chief among these advances is determining the molecular identity of defensive chemicals and the roles they play in shaping interactions between individuals. Some progress has been made in deciphering the molecular, cellular, and systems level mechanisms underlying these interactions, as well as how these interactions can lead to structuring of communities and even ecosystems. The neuroecological approach unifies practices and principles from these diverse disciplines and at all scales as it attempts to explain in a single conceptual framework the abundances of organisms and the distributions of species within natural habitats. This article explores the neuroecology of chemical defenses with a focus on aquatic organisms and environments. We review the concept of molecules of keystone significance, including examples of how saxitoxin and tetrodotoxin can shape the organization and dynamics of marine and riparian communities, respectively. We also describe the current status and future directions of a topic of interest to our research group-the use of ink by marine molluscs, especially sea hares, in their defense. We describe a diversity of molecules and mechanisms mediating the protective effects of sea hares' ink, including use as chemical defenses against predators and as alarm cues toward conspecifics, and postulate that some defensive molecules may function as molecules of keystone significance. Finally, we propose future directions for studying the neuroecology of the chemical defenses of sea hares and their molluscan relatives, the cephalopods.Integrative and Comparative Biology 06/2011; 51(5):771-80. · 3.02 Impact Factor
The purple pigment aplysioviolin in sea hare ink deters predatory blue crabs
through their chemical senses
Michiya Kamioa,b,*, Tiphani V. Grimesa, Melissa H. Hutchinsa, Robyn van Dama, Charles D. Derbya
aNeuroscience Institute and Department of Biology, Georgia State University, Atlanta
bDepartment of Ocean Science, Tokyo University of Marine Science and Technology
a r t i c l e i n f o
Received 30 October 2009
Initial acceptance 17 December 2009
Final acceptance 25 March 2010
Available online 11 May 2010
MS. number: A09-00708
Sea hares release an ink secretion composed of purple ink and white opaline as a potential chemical
defence against predators. The aim of our study was to identify deterrent molecules in the ink of Aplysia
californica against an allopatric generalist crustacean predator, the blue crab Callinectes sapidus, and to
define the mechanisms of action of the deterrents against crabs. We used two behavioural assays,
a squirting assay and an ingestion assay, to show that ink is highly effective and that opaline is
moderately effective in suppressing feeding of crabs. Results with reversibly blinded crabs demonstrate
that the deterrence is mediated through the crabs’ chemical senses. We used bioassay-guided frac-
tionation to identify the purple molecules aplysioviolin and phycoerythrobilin as a major and minor
deterrent, respectively, in ink against crabs. These molecules derive from a light-harvesting protein in the
photosynthetic system of dietary algae. This is the first demonstration of an animal converting
a photosynthetic pigment into a chemical deterrent. Mixing opaline and ink enzymatically produces
hydrogen peroxide, which also functions as a chemical deterrent against crabs. Our results and those of
other studies show that sea hares use a diversity of molecules in their skin, mucus and ink secretion to
chemically defend themselves against their potential predators. Aplysioviolin, phycoerythrobilin and
hydrogen peroxide also exist in ink secretion of Aplysia dactylomela, a sea hare sympatric to blue crabs,
and thus we posit that these molecules are potentially effective in ecologically relevant predatoreprey
interactions and need to be scrutinized in more ecologically relevant experiments.
? 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Many species with low mobility, from plants to animals, defend
themselves from attack by consumers using constitutive chemical
defences that cover their body surface or are embedded in their
tissues (McClintock & Baker 2001; Clark et al. 2005; Paul & Ritson-
Williams 2008; Wink 2008). In addition, many organisms also
respond to predatory attacks using activated chemical defences in
their tissues (Hadacek 2002; Wittstock & Gershenzon 2002; Van
Alstyne & Houser 2003; Thoms & Schupp 2008; Walling 2009) or
by releasing chemicals tothe external environment around them to
deter predators (Eisner & Aneshansley 1999; Wood 1999; Shiomi
et al. 2001; Williams & Gong 2007). Many of these released mole-
cules probably affect the chemosensory systems of consumers, as
has been experimentally demonstrated in some cases (Kicklighter
et al. 2005; Nusnbaum & Derby 2010). Some of these molecules
are pigmented (i.e. absorb light in the visible range of predators)
and thus might act as defences through the visual modality of
predators. Such ‘ink’ can protect an animal by acting as a smoke
screen behind which it can hide from a predator, as a decoy that
attracts the attention of the predator, or as a stimulus that startles
the predator (Derby 2007; Wood et al. 2008). Thus, pigmented
molecules have the potential to affect both the visual and chemical
senses of the releaser’s enemies. Animals can sequester chemical
deterrents from their food and modify them (Garson 2001;
Hadacek 2002; Clark et al. 2005), or synthesize them de novo
(Garson 2001; Cimino & Ghiselin 2009).
Sea hares (Opisthobranchia: Anaspidea) live in marine benthic
communitieswhere they have
concentrating on red and green algae (Carefoot 1987). They are
mobile but slowand have a soft body without a shell for protection.
Consequently, they chemically defend themselves against a range
of predators by sequestering secondary metabolites from their
dietary red algae and mobilizing them into their skin and digestive
glands (Paul & Pennings 1991; Ginsburg & Paul 2001; Kamiya et al.
2006). In addition to these constitutive chemical defences in their
skin and tissues, sea hares also have a behaviourally activated
chemical defence, ink secretion, which is released when they are
attacked by predators (Nolen et al. 1995; Johnson & Willows 1999;
Pennings et al.1999). Ink secretion is composed of two co-released
glandular products: ink, which is a dense, dark purple product
released from the ink gland, and opaline, which is a translucent,
relatively specialized diets,
* Correspondence: M. Kamio, Department of Ocean Science, Tokyo University of
Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
E-mail address: firstname.lastname@example.org (M. Kamio).
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/anbehav
0003-3472/$38.00 ? 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Animal Behaviour 80 (2010) 89e100
whitish product released from the opaline gland. Ink secretion of
the sea hare Aplysia dactylomela suppresses feeding of laughing
gulls and blue crabs (DiMatteo 1981, 1982), ink secretion from
Dolabella auricularia is unpalatable to reef fishes (Pennings et al.
1999), and ink secretion from Aplysia californica is a deterrent of
sea anemones, spiny lobsters and a variety of fishes (DiMatteo
1981; Nolen et al. 1995; Kicklighter et al. 2005; Kicklighter &
Derby 2006; Aggio & Derby 2008; Sheybani et al. 2009;
Nusnbaum & Derby 2010, in press). However, in only a few cases
have the molecules responsible for the chemical deterrence of ink
been identified (Kicklighter et al. 2005; Aggio & Derby 2008). One
case is the amino acid components of ink and opaline, which
function as a deterrent against spiny lobsters. Ink and opaline
contain millimolar concentrations of amino acids, which by
themselves are highly attractive to many predators including
crustaceans and fishes (Derby & Sorensen 2008). Because of its
stimulatory amino acids, ink secretion itself can sometimes over-
come unpalatablemolecules in ink and stimulate appetitivefeeding
responses of predatory spiny lobsters, a process called phag-
omimicry (Kicklighter et al. 2005). Another case is molecules
generated by escapin. Escapin is an L-amino acid in ink (Yang et al.
2005). When ink and opaline are co-secreted, escapin oxidizes L-
lysine, which is present in high millimolar amounts in opaline
(Johnson et al. 2006). This reaction produces three molecules:
hydrogenperoxide, ammonia and the alpha-keto acid of lysine. The
alpha-keto acid exists as an equilibrium mixture of imine, enamine
and other forms in an aqueous solution (called ‘escapin interme-
diate products’), and these compounds react with hydrogen
peroxide to produce another set of compounds (called ‘escapin end
products’) (Kamio et al. 2009). Products of the escapin pathway are
deterrent to spiny lobsters (Aggio & Derby 2008) and wrasses
(Nusnbaum & Derby, in press). Despite these examples, and despite
abundant knowledge that ink secretion is unpalatable to many
animals, no deterrent molecules in ink secretion itself have been
identified through bioassay-guided fractionation.
The overall goal of our study was to find chemosensory deter-
rent molecules in ink or opaline using the sea hare Aplysia
californica and the predatory blue crab Callinectes sapidus as animal
models. Towards this goal, we had several aims in our experiments.
The first aim was to determine whether sea hares use ink and
opaline as a phagomimetic defence oras a deterrent defence, and to
assess the relative importance of ink and opaline. The second aim
was to identify the deterrent molecules in the ink. The third aim
was to identify which of the predators’ sensory modalities is
affected by these molecules. We also tested the deterrence of the
enzymatic reaction products that are produced by co-secretion of
ink and opaline.
We used blue crabs in our study because they are generalist
laboratory and which use chemoreception in many aspects of their
behaviour including feeding (Pearson & Olla 1977; Rittschof 1992;
Weissburg & Zimmer-Faust 1994). Sea hares typically release ink
when vigorously attacked by predatory blue crabs presented with
small juvenile (ca. 1 g) or adult (200e300 g) sea hares (M. Kamio,
unpublished observations). Sea hares can release ink before or after
being bitten or pinched by crabs, and sea hares can control the
direction of inking towards the attacking predator (Walters &
Erickson 1986). During encounters in which ink is released, ink
can stimulate different sensory organs on the predator’s body. For
crustaceans such as blue crabs, these sensors can include the first
and second antennae, the mouthparts, or the legs, which are major
chemosensory organs that control different aspects of feeding
behaviour (Derbyet al. 2001). Those sensors might be stimulated in
the presence or absence of body fluids released from damaged sea
hares, defensive ink secretions, or when the predator has the sea
hare in its mouth. The inking often stops blue crabs’ attacking
behaviour and gives sea hares a chance to escape. We used ink from
the sea hare Aplysia californica, which is allopatric to blue crabs,
rather than from the blue crab’s sympatric sea hare, Aplysia dacty-
lomela, because A. californica is better studied and readily available.
All animals used in our study were collected from the field.
Mature male blue crabs, Callinectes sapidus, with carapace length of
10e12 cm were obtained from Gulf Specimen Marine Lab (Panacea,
FL, U.S.A.) or commercial fisherman in St Augustine, Florida,
maintained in our laboratory in aquaria with recirculating, filtered
and aerated artificial sea water (Instant Ocean, Aquarium Systems,
Mentor, OH, U.S.A.) at 20?C, and fed squid and shrimp. Adult
(200e300 g) sea hares, Aplysia californica, were collected by
Marinus Scientific (Garden Grove, CA, U.S.A.), and adults of Aplysia
dactylomela were obtained from the Keys Marine Laboratory
(Layton/Long Key, FL). Sea hares were used immediately upon their
arrival in the laboratory. Animal care and experiments were per-
formed within university regulations and national guidelines. Only
animals that ate pieces of food or responded appetitively to food-
related chemical stimuli were used in our experiments.
Chemical Stimuli and Reagents
To collect ink and opaline, we dissected ink and opaline glands
from anaesthetized sea hares immediately upon their arrival in our
laboratory. Ink and opaline glands were frozen at ?80?C until used.
Ink was collected bygently squeezing dissected ink glands in a petri
dish with the blunt end of a scalpel handle. Opaline was collected
by centrifuging opaline glands at 30 000 ? g for 1 h at 4?C. Shrimp
juice was prepared by homogenizing 1 g of fresh shrimp abdominal
muscle in 50 ml of sea water. Sea water was either artificial sea
water (Instant Ocean?) or filtered sea water (pumped and filtered
from sea water near the Whitney Laboratory, University of Florida).
Freeze-dried shrimp was made from frozen shrimp using a lyophi-
lizer.Pieces of freeze-dried
(90e130 mg dry mass) were used in feeding experiments. Bilirubin
(to determine structureeactivity relationships), sodium alginate
and calcium chloride (to prepare food pellets) were purchased from
SigmaeAldrich (St Louis, MO, U.S.A.). The intermediate products of
escapin’s oxidation of L-lysine (escapin intermediate products, or
EIP) were prepared by incubating L-lysine monohydrochloride,
escapin (purified from ink) and catalase in double distilled water as
described in Kamio et al. (2009).
shrimp of ca.5 ? 5 ?10 mm
We tested substances for their effects on blue crabs in two
general categories of assays: an assay that measures the chemical’s
ability to stimulate feeding behaviour and thus serve in defence as
a phagomimic; and assays that measure the chemical’s ability to
inhibit appetitive responses to food odours and ingestion of food.
Then, we used bioassay-guided fraction, based on an inges-
behavioural responses of crabs, to identify components in ink
responsible for its deterrent effects. We also used the inges-
tioneinhibition assay to test the activity of escapin products. The
effect of the deterrents on the chemical sense of blue crabs was
confirmed using eye caps to reversibly deprive animals of visual
M. Kamio et al. / Animal Behaviour 80 (2010) 89e100
Test of ink and opaline as phagomimics: sponge assay
Ink itself stimulates feeding responses from spiny lobsters and
in so doing functions as a phagomimetic defence (Kicklighter et al.
2005). In this experiment, we examined whether ink also activates
feeding behaviour of blue crabs. Crabs were placed in a plastic tank
(20 ? 35 ? 20 cm) filled with sea water and acclimated for 30 min.
Only crabs that ate a small piece of shrimp were tested, and each
crab was tested with a series of four stimuli (ink, opaline, shrimp
juice, and sea water) presented once each in random order. Stimuli
were presented to a crab by spotting 100 ml of stimulus onto the
edge of a 1 ?1.5 ? 5 cm piece of plastic sponge (Beauty Wedges?,
Apothecary Products, Inc., Minneapolis, MN, U.S.A.) and using tongs
to move the spotted edge of the sponge towards the crab until it
contacted the crab’s chela. Shrimp juice was used as a positive
control and sea water as a negative control. Each trial lasted 1 min,
which was sufficient time forall crabs torespond.To protectagainst
observer bias, the experimenter presented stimuli blind to their
identity; however, because the colour and consistency of ink and
opaline differ, it was not possible to completely hide their identity
from the experimenter. The recorded behavioural responses were:
0, push away: push the sponge away from the body; 1, take to
mouth: take the sponge to mouthparts but do not bite it; 2, single
bite: bite the sponge and tear off a piece; and 3, multiple bites: take
more than one bite from the sponge. These four behaviours were
graded from 0 to 3 in statistical analysis, and labelled as appetitive
(single bite, multiple bites, take to mouth), or rejection (push
away). Ten crabs were tested with the series of stimuli. We tested
for differences among groups using Friedman ANOVA, and used
two-tailed Wilcoxon signed-ranks tests with Bonferroni correction
(a ¼ 0.0083) as post hoc tests to identify differences between
Effect of ink and opaline on appetitive responses to food odours:
To determine whether ink affects the response to a food odour,
we used a pipette to squirt stimuli towards the mouthpart of the
crab in shallow water. Crabs were acclimated for 30 min in glass
aquaria (50 ? 25 ? 30 cm) in which the water level was just above
the cephalothorax of the crab. Crabs usually used their chelae to
pick up their food, so when shrimp juice was introduced at the
water surface just above the crab, the crab typically searched for
food by extending its chelae forward and tried to grab and pull it to
its mouth evenwithout visual stimuli. Crabs showed this behaviour
to a piece of food introduced into the aquarium, but not to sea
water. We interpret this to be food-seeking behaviour. If a crab did
not show food seeking to shrimp juice or shrimp itself, it was not
included in the assay. Only crabs showing this food-seeking
behaviour were used for the experiment and each crab was tested
one each with a series of stimuli. To evaluate the ability of ink or
opaline to suppress the food-seeking behaviour to shrimp juice, we
presented crabs with shrimp juice released from a tube powered by
a peristaltic pump at a flow rate of 5 ml/min, which caused all crabs
used in the analysis to perform food-seeking behaviour. While
stimulated with shrimp juice for 10e15 s, crabs were then also
presented with 100 ml aliquots of other stimuli, by squirting each
stimulus with a hand-held pipette towards the mouth of the crab
within 1 s. The stimuli tested were sea water (negative control)
followed by either full strength opaline, full strength ink, or full
strength aplysioviolin (APV). We scored the behaviour of crabs
according to whether they continued or ceased food-seeking
behaviour in the presence of the stimuli. Cessation of food-seeking
behaviour occurred within a few seconds after introducing an
aversive stimulus. Ten crabs were tested with seawater and opaline
sequentially, 12 crabs were tested with sea water and ink sequen-
tially, and 10 crabs were sequentially tested with sea water and
purified APV. Data are expressed as the percentage of crabs (out of
10e12 tested) that showed food-seeking behaviour, and analysed
with a one-tailed McNemar’s test to determine whether the
presentation of ink, opaline or APV during the presentation of
shrimp juice caused significantly fewer crabs to show food-seeking
behaviour compared to the presentation of sea water (a ¼ 0.05).
Because peaks in the absorbance spectrum of ink (Nusnbaum &
Derby2010; this study) overlapthose of the primary visual pigment
of blue crabs (Cronin & Forward 1988) whereas those of the other
stimuli do not, our experimental design could not eliminate the
possibility that crabs might use vision to respond. To confirm the
chemosensory effect of ink, we reversibly blinded the crabs by
placing ‘eyes caps’, made from heat-shrink tubing, over their eyes.
The experimental protocol, performed on 10 crabs, was the same as
described above with the exception that we did not test opaline.
Effect of ink, opaline and products of the escapin pathway on
ingestion of food: dry shrimp assay
We spotted freeze-dried shrimp with 100 ml of sea water
(untreated) or with 100 ml of either full strength ink or opaline (test
stimuli treated) and fed one piece of shrimp from the control
treatment and one piece of ink treatment to each of 10 crabs in
random order. Nine crabs were tested for opaline treatment. The
difference in weight between the two pieces of shrimp fed to each
crab was less than 8%. We assessed two dependent measures for
each trial: (1) whether the crab ate all of the shrimp (‘ate’), ate none
of the shrimp (‘rejected’), or manipulated the shrimp in mouthparts
but released the shrimp without eating it (‘bit and peeled’); and (2)
for crabs that did not reject the shrimp (i.e. those that ‘ate’ or ‘bit
and peeled’ the shrimp), how long the crab held the shrimp in the
mouth before either ingesting the entire shrimp (for crabs that
‘ate’) or completing bite and peel. The first three behaviours were
graded from 0 to 2 for statistical analysis (0 ¼ ate, 1 ¼ bit and
peeled, 2 ¼ rejected). Behaviour of animals was observed for 300 s.
The dependent measures were analysed with a one-tailed
Wilcoxon signed-ranks test (a ¼ 0.05), to determine whether crabs
responded differently to ink or opaline-treated and untreated
Bioassay-guided Fractionation to Identify Bioactive Molecules
To identify deterrent molecules, we performed bioassay-guided
fractionation of ink. The bioassay was an ingestion assay, in which
chemicals were added to food (artificial food pellets flavoured with
shrimp powder) and their effects on ingestion were quantified. We
used an artificial food because small quantities of chemicals could
be homogeneously incorporated into it and crabs readilyeat it (Hay
et al. 1998b). The amount of shrimp powder added to the pellets
was selected to be the least amount that caused most crabs to eat
the pellets. Control pellets were prepared by mixing 0.1 g of freeze-
dried shrimp powder with 1 ml of 2% sodium alginate in deionized
water. Experimental pellets were prepared similarly except ink
fractions orother stimuli were added to them. Each ink fractionwas
tested at three times its concentration in full strength ink to
increase the efficiency of the bioassay used to purify active
compounds. Water-insoluble stimuli were solubilized in 40%
ethanol, which was also used in the control food. The final
concentration of ethanol in test and control pellets was 5%. Pellets
were made by extruding from a 1 ml syringe the alginate/shrimp/
chemical mixture into 0.25 M CaCl2, which caused the mixture to
solidify into about 15 pellets, each 27e64 mm3. Each crab was held
in a glass aquarium (50 ? 25 ? 30 cm) and fed a piece of shrimp 3 h
before the experiment, and only crabs that ate a control pellet
M. Kamio et al. / Animal Behaviour 80 (2010) 89e100
presented at the start of the experiment were used in the assay.
Each trial lasted 60 s, which was a sufficient time for a crab to either
eat or reject a pellet. Satiation was tested by giving a control pellet
after each test pellet, and only if the crab ate the control pellet was
the data for the test pellet included in the analysis. Each crab was
tested only once for a series of stimuli.
The frequency of rejection of food-flavoured pellets containing
each fraction was compared to rejection of a control pellet (i.e.
food-flavoured pellets containing sea water), using a one-tailed
McNemar’s test (a ¼ 0.05) to determine whether the addition of
each fraction increased the frequency of rejection.
To fractionate ink for testing in the bioassay, 159 ml of ink was
acquired from 117 ink glands. Ink was diluted with methanol to
a final concentration of 80% methanol and centrifuged at 1000 ? g
for 10 min to precipitate proteins and other insoluble macromole-
cules and salt. The supernatant, which was purple, was dried using
a rotary evaporator, solubilized in 40% methanol, and absorbed
onto a column with Diaion HP20SS gel (Mitsubishi Chemical USA,
Inc.) for separation on the basis of their polarity. Compounds were
sequentially eluted from the column with 40, 60, 80 and 100%
aqueous methanol and fractionated. Fractions with similar spots on
thin-layer chromatograms (silica gel, solvent of 4:1:2 butanol:
acetic acid:water) were combined to yield five fractions (F1eF5),
whose activities were tested using the behavioural bioassay. One of
the resultant active fractions (F4) was separated according to
polarity using reversed-phase HPLC, with a Phenomenex Luna C18
(2) column (25 ? 2 cm) using a solvent system containing 100 mM
NaClO4, isocratic 80% methanol elution for 15 min followed by
80e100% methanol linear gradient for 10 min and 100% methanol
isocratic elusion for 10 min. Flow rate was 8 ml/min. We bioassayed
the activity of each of the resultant four peaks (P1eP4). P3, which
was the peak with the most bioactivity and the highest abundance,
was further separated using a Phenomenex Luna C18 (2) column
(25 ?1 cm) with a different solvent system (isocratic 5:2 iso-
propanol:water at 0.5 ml/min) and by monitoring the elusion at
280 nm. This yielded three peaks: P3-1, P3-2 and P3-3. We tested
these peaks, as well as the other fractions combined (¼‘other’),
singly for bioactivity using the food-flavoured alginate pellet
ingestion assay. We identified the structure of the bioactive mole-
cule at its concentration in full strength ink (i.e. P3-2) using nuclear
magnetic resonance and mass spectrometry. The details of the
molecular identification with spectral data, showing that P3-2 is
APV, and that another minor bioactive molecule is phycoery-
throbilin (PEB), which was found in other separation on the other
batch of ink, will be published separately (M. Kamio, L. Nguyen,
S. Yaldiz & C. D. Derby, unpublished data).
Contribution of Aplysioviolin, Phycoerythrobilin and Other
Components to the Total Deterrent Activity of Ink
To test the contribution of APV, PEB and other components of
ink, both singly and in combination, towards the total deterrent
activity of ink, we fractionated ink. The isolation scheme was
simplified for this experiment compared to the one above. We
diluted 4 ml of ink with 16 ml of methanol and centrifuged the
solution to remove precipitated proteins and other large molecules.
The precipitate, which was dark purple, was extracted with
methanol repeatedly until it became white. The resultant methanol
supernatant, which was also purple, was dried using a rotary
evaporator, then solubilized in 40% methanol and absorbed onto
a column with Diaion HP20SS gel. The column was eluted
sequentially with 40% methanol and 100% methanol to produce
two fractions. We further fractionated the 100% methanol fraction
via reversed-phase HPLC using a Waters 2487 Detector and
a Waters 1525 Pump equipped with a Phenomenex Luna C18 (2),
5 mm, 10 ? 250 mm column. For separation, we used a solvent
system of 40e45% acetonitrile linear gradient containing 0.1%
trifluoroacetic acid for 30 min, at a flow rate of 2 ml/min, and we
monitored elution at 534 nm. This separation of the 100% methanol
elution resulted in three fractions: one containing pure APV, one
containing pure PEB, and a third containing all other fractions
combined (¼‘other’). These three fractions and the 40% methanol
elution (¼40 M) were dried, weighed and tested individuallyand in
combination in behavioural bioassays. All fractions were tested at
10%,1% and 0.1% equivalent concentrations of natural ink using the
food-flavoured alginate pellet ingestion assay. Fractions were
solubilized with ethanol and paired with a control containing the
same amount of ethanol, with a final concentration of 5% in food
To determine the structureeactivity relationship of the deter-
rent compounds, we compared the bioactivity of bilirubin, APV and
PEB, which are all linear tetrapyrroles, at equal concentration
(2.5 mg/ml), which is 10% ink equivalent concentration for APV.
Activity of Aplysioviolin in Squirting Assay
Since APV was purified by bioassay-guided fractionation using
the ingestion assay, we also evaluated the deterrence of APV in
another assay, the squirting assay. We tested purified APV in the
squirting assay, as described above, with blinded crabs at
a concentration of 1 mg/ml in 10% ethanol in sea water. We used
10% ethanol in sea water as a control.
Deterrence of Escapin Products
We tested products from the escapin pathway at full strength
concentrations (145 mM) in the freeze-dried shrimp assay, as
described above, except that crabs were observed for 120 s (not
300 s). The stimuli tested were: lysine (K), hydrogen peroxide (HP),
ammonium (NH4), escapin intermediate product of lysine (EIP),
escapin end product of lysine (EEP), EIP þ HP, and HP þ NH4.
We used the software program R (v2.8.1) for statistical analysis
(the R Foundation for Statistical Computing, Vienna, Austria).
Ink and Opaline Are Not Phagomimics for Blue Crabs
A sponge spotted with shrimp juice, the positive control, evoked
appetitive behaviours from all 10 crabs, with six crabs showing
‘take to mouth’ and four showing ‘multiple bites’ (Fig. 1). A sponge
spotted with sea water, the negative control, evoked rejection
(push away) from most crabs (N ¼ 9) and appetitive responses
(take to mouth) from one. A sponge spotted with full strength ink
evoked rejection (push away) from most crabs (N ¼ 7) and appe-
titive responses from three crabs (take to mouth,1 crab; single bite,
2 crabs). A sponge spotted with full strength opaline evoked push
away (8 crabs), take to mouth (1 crab) and single bite (1 crab). Crabs
always spit out any piece of sponge that they bit from the sponge.
Overall, there was a statistically significant difference in the
responses evoked by these four stimuli (Friedman test: c3
N ¼ 10, P < 0.01), with shrimp juice being different from ink,
opaline and sea water (Wilcoxon signed-ranks test: P < 0.0083),
and all others not being different from each other (P > 0.0083).
Thus, neither opaline nor ink evoked statistically significant levels
of appetitive feeding behaviours.
M. Kamio et al. / Animal Behaviour 80 (2010) 89e100
Squirting Assay: Crabs’ Food-seeking Responses to Food Odours Are
Strongly Inhibited by Ink and Moderately Inhibited by Opaline
Crabs with a normal visual system (i.e. without eye caps)
showed food-seeking behaviour towards a tube that continuously
released a food odour, shrimp juice (‘shrimp juice þ sea water’;
Fig. 2a). The crabs’ response to shrimp juice was differentially
affected by the co-release of ink or opaline. Response to shrimp
juice was significantly higher in sea water than in ink (one-tailed
McNemar’s test: c13
McNemar’s test: c1
higher in opaline than in ink (two-tailed Fisher’s exact test:
P ¼ 0.032). Crabs that were blinded with eye caps performed
similarly. All 10 crabs showed food-seeking behaviour to shrimp
juice with sea water, whereas 9 of 10 crabs stopped food-seeking
behaviour to shrimp juice in ink (Fig. 2b); this effect was statisti-
cally significant (one-tailed McNemar’s test: c1
Thus, ink inhibited the crabs’ food-seeking response to shrimp
juice, whether the crabs could see or not.
2¼ 10.08, P ¼ 0.0005) or opaline (one-tailed
2¼ 3.2, P ¼ 0.036). Response to shrimp juice was
2¼ 7.11, P ¼ 0.004).
Dry Shrimp Assay: Ingestion of Food is Strongly Inhibited by Ink and
Moderately Inhibited by Opaline
Freeze-dried shrimp spotted with sea water was completely
ingested by all 10 crabs (Fig. 2c). In contrast, freeze-dried shrimp
spotted with full strength ink was either rejected (N ¼ 2 crabs), or
bitten and peeled but not eaten (N ¼ 8 crabs). The frequencies of
these behaviours to freeze-dried shrimp spotted with sea water or
ink differed significantly (one-tailed Wilcoxon signed-ranks test:
T ¼ 0, N ¼ 10, P ¼ 0.002; Fig. 2c). When crabs did not reject shrimp
outright, shrimp spotted with ink were held in the crab’s mouth
spotted shrimp were bitten and peeled first, then rejected, whereas
shrimp spotted with sea water were eaten immediately (one-tailed
Wilcoxon signed-ranks test: T ¼ 0, N ¼ 10, P ¼ 0.001; Fig. 2d).
Adding opaline to dry shrimp did not significantly affect the
frequency of ingestive behaviours to the shrimp. All but one crab
ate freeze-dried shrimp spotted with opaline, which was not
different from the number that ate shrimp spotted with sea water
(one-tailed Wilcoxon signed-ranks test: T ¼ 22.5, N ¼ 9, P > 0.05;
Fig. 2c). However, handling times for shrimp spotted with opaline
were significantly longer than those for shrimp spotted with sea
water (one-tailed Wilcoxon signed-ranks test: T ¼ 2, N ¼ 9,
P ¼ 0.008; Fig. 2d).
Bioassay-guided Fractionation Identifies Aplysioviolin as a Deterrent
Compound in Ink
Separation of ink using an HP20SS column produced four frac-
tions, F1eF4, two of which (F3 and F4) evoked significant rejection
by blue crabs at three times the ink equivalent concentration tested
in the ingestion assay using food-flavoured alginate pellets (one-
tailed McNemar’s test: P < 0.05; Fig. 3a). We focused on F4 since it
produced a prominent peak on the chromatogram, whereas F3 did
not. Further separation of F4 according to polarity using reversed-
phase HPLC with NaClO4in aqueous methanol solvent produced
four peaks (P1eP4), only one of which (P3) showed significant
rejection by blue crabs in the alginate pellet ingestion assay (one-
tailed McNemar’s test: P < 0.05; Fig. 3b). Further separation of P3
using reversed-phase HPLC with a solvent of 5:2 isopropanol:water
yielded three peaks (P3-1, P3-2, P3-3), only one of which (P3-2)
evoked significant rejection by blue crab in the pellet ingestion
assay (one-tailed McNemar’s test: P < 0.05; Fig. 3c). P3-2 contained
a pure compound, which we determined to be aplysioviolin (APV)
(Fig. 4a) based on nuclear magnetic resonance, mass spectrometry
and UV spectroscopy. Details of spectral data and identification of
the structure of APV will be published elsewhere (M. Kamio,
L. Nguyen, S. Yaldiz & C. D. Derby, unpublished data). In another
separation on another batch of ink, phycoerythrobilin (PEB)
(Fig. 4a) was found to be an active component.
Aplysioviolin is a Major Deterrent in Ink but Not the Sole Deterrent
To evaluate the relative contributions of APV and PEB to the
bioactivity of ink, four fractions were obtained from HP20SS and
HPLC for behavioural testing individually and in combination.
These were, in order of polarity from relatively low to high: (1) the
40% MeOH fraction (¼40 M); (2) all fractions besides the other
three (¼OT); (3) phycoerythrobilin (¼PEB); and (4) aplysioviolin
(¼APV) (Fig. 4a). These four fractions constituted 80%, 7.5%, 2.5%
and 10% of the total dry mass, respectively (Fig. 4b).
When we tested the four fractions individually at 10% of the
ink equivalent concentration in the food-flavoured alginate pellet
ingestion bioassay, only APV showed significant deterrent activity,
causing 60% of crabs to reject food-flavoured alginate pellets (one-
tailed McNemar’s test: P < 0.05; Fig. 4d). At 1% and 0.1% ink
equivalents, APV showed low and statistically nonsignificant
deterrency (20% and 10% rejection, respectively; P > 0.05). The
other three fractions (PEB, OT and 40 M) caused 0e10% rejection
over the 10e0.1% ink equivalent range (P > 0.05). The positive
control (ink) caused rejection of 90%, 20% and 10% of crabs at 10%,
1% and 0.1%, respectively, with only the 10% ink evoking a signif-
icant response (P < 0.05), and the 0.1% full strength and the
negative control (sea water) not evoking any rejection of food
(P > 0.05).
When we tested the four fractions in combinations, it became
apparent that fractions besides APV had deterrent activity (Fig. 4e).
At 10% ink equivalents, even combinations that did not contain APV
(i.e. OT þ 40 M and PEB þ OT þ 40 M) evoked significant deter-
rence (i.e. 63.6% and 50% rejection, respectively; one-tailed
McNemar’s test: P < 0.05), similar to the mixture of all four frac-
tions (70%). No combinations were significantly active at 1% and
0.1% ink equivalents (P > 0.05).
When we tested the individual pure compounds APV, PEB and
bilirubin at equivalent concentrations (6.25 mg/ml; the concen-
tration of APV at 10% ink equivalent), APV and PEB had similar and
statistically significant deterrency (60% and 70% rejection, respec-
tively; one-tailed McNemar’s test: P < 0.05), while bilirubin
showed lower and statistically nonsignificant activity (30%;
P > 0.05; Fig. 4c).
% Crabs showing appetitive
or rejection behaviour
Take to mouth
Opaline SW Ink
N = 10
Figure 1. Effect of ink and opaline on blue crab feeding behaviour in the sponge assay.
Crabs’ responses to 100 ml of ink, opaline, shrimp juice (positive control) and sea water
(SW;negativecontrol)presentedonpiecesofspongeinrandomorder.*P < 0.01(overall
difference in responses to the four stimuli; Friedman test); **P < 0.0083 (differences
between pairs of stimuli; Wilcoxon signed-ranks test with Bonferroni correction).
M. Kamio et al. / Animal Behaviour 80 (2010) 89e100
Purified Aplysioviolin Suppresses Food-seeking Behaviour of Blue
Crabs in Squirting Assay
All 10 crabs that were reversibly blinded showed food-seeking
behaviour with shrimp juice þ sea water (Fig. 5). When APV was
squirted with shrimp juice, 7 of 10 crabs stopped food seeking.
Responses to APV differed significantly from those to sea water
(one-tailed McNemar’s test: c1
2¼ 5.1429, P ¼ 0.0115). Thus, purified
Concentrations of Aplysioviolin and Phycoerythrobilin in Aplysia
californica and Aplysia dactylomela
Concentrations of APV and PEB were measured in ink fromwild-
caught sea hares, Aplysia californica and Aplysia dactylomela. In
both, APV was always in higher concentration than PEB in all
measurements, although the difference was not statistically
significant (two-tailed paired t test: t2¼1.9239, N ¼ 3, P ¼ 0.19 for
A. dactylomela; Fig. 6). Concentrations of APV and PEB in ink were
27 mg/ml and 3 mg/ml, respectively, for A. californica, and 2.4 mg/
ml and 0.7 mg/ml, respectively, for A. dactylomela. The APV:PEB
ratio was 9.0 for A. californica ink and 3.4 for A. dactylomela ink.
Escapin Products Suppress Feeding Behaviour of Blue Crabs
We evaluated escapin products (Fig. 7a) for effects on feeding
behaviour of blue crabs using the freeze-dried shrimp ingestion
assay. All crabs took all shrimp pieces into their mouths (i.e. no
‘rejects’). Shrimp spotted with sea water, lysine or EEP were
completely ingested by all 10 crabs (Fig. 7b). In contrast, shrimp
% Crabs showing food−seeking behaviour
N=10 or 12
Handling time (s)
% Crabs showing food−seeking behaviour
Dry shrimp assay
% Crabs showing each behaviour
Figure 2. Mean percentage of (a) intact and (b) blinded blue crabs showing food-seeking behaviour in the squirting assay. (c) Mean percentage of blue crabs that ate the shrimp
(eat), bit and peeled but did not eat the shrimp (bite & peel), or rejected the shrimp (reject) in the dry shrimp assay. (d) Blue crab handling time of shrimp in the dry shrimp assay.
Values are medians ? interquartile ranges. SW ¼ sea water. *P < 0.05; **P < 0.005; ***P < 0.0005.
M. Kamio et al. / Animal Behaviour 80 (2010) 89e100
spotted with HP, NH4, EIP, EIP þ HP, or HP þ NH4were bitten and
peeled but not ingested. This was the case for five crabs with
shrimp spotted with HP þ NH4, four crabs with HP, four crabs with
EIP þ HP, one crabwith NH4, and one crabwith EIP. The frequencies
of these behaviours to the sea water control differed significantly
from those to HP (one-tailed Wilcoxon signed-ranks test: T ¼ 10.5,
N ¼ 10,
P < 0.05), EIP þ HP(T ¼ 10.5,
N ¼ 10,
P < 0.05)and
HP þ NH4 (T ¼ 5.5, N ¼ 10, P < 0.05; Fig. 7b), but did not differ
significantly from those to the other stimuli. Shrimp spotted with
lysine, HP, EIP, EIP þ HP or HP þ NH4were held in the crab’s mouth
significantly longer than were shrimp spotted with sea water
(Wilcoxon signed-ranks test: lysine: T ¼ 10, N ¼ 10, P < 0.05; HP:
T ¼ 5, N ¼ 8, P < 0.01; EIP: T ¼ 8, N ¼ 10, P < 0.05; EIP þ HP: T ¼ 1,
N ¼ 8, P < 0.01; HP þ NH4: T ¼ 5, N ¼ 6, P < 0.01; Fig. 7c), because
crabs took longer to ingest shrimp (lysine), or to bite, peel and
reject shrimp (HP, EIP, EIP þ HP and HP þ NH4).
Our results show that ink secretion of A. californica functions as
a deterrent against blue crabs. Ink secretion affects two phases of
suppresses food-seeking behaviour, responses to food odours and
ingestion of food once taken to the mouth. Of the two glandular
contributors to ink secretion’s deterrence, ink (the purple secretion
from the ink gland) is more effective than opaline (the white,
mucous secretion from the opaline gland). A major component
contributing to the deterrency of ink is the coloured molecule
aplysioviolin. Aplysioviolin is derived from photosynthetic proteins
in dietary red algae (Chapman & Fox 1969; Coelho et al. 1998;
Prince et al. 1998; Prince & Johnson 2006; M. Kamio, L. Nguyen,
S. Yaldiz & C. D. Derby, unpublished data), and thus sea hares
convert photosynthetic proteins to chemical deterrents that affect
the chemosensory system of blue crab. This is the first demon-
stration of an animal converting plant photosynthetic molecules
into an antipredatory chemical defence. Our results are from
laboratory experiments using an allopatric model predatoreprey
system, and thus, whether and how these mechanisms operate in
the field to improve sea hare survival remains to be experimentally
tested. Other factors besides ink undoubtedly contribute to the
survival of sea hares in the field. Nevertheless, our study indicates
that aplysioviolin might be an important predator deterrent used
by A. californica and other sea hare species in an ecological context.
Ink Secretion Deters Blue Crabs by Acting on their Chemical Senses
and is Potentially One of the Defensive Systems of Sea Hares Against
Our results show that both glandular components of ink
secretion, ink and opaline, suppress behaviour of crabs towards
food odours or food itself, and thatthe effectof ink is much stronger
than that of opaline. Either ink or opaline inhibited food-seeking
behaviour when squirted at the anterior end of crabs that were
simultaneously stimulated with food odour (Fig. 2a, b), or when
incorporated into food (food-flavoured alginate pellets: Fig. 5d; dry
shrimp: Fig. 2c, d). In addition, neither ink nor opaline presented to
blue crabs in the absence of food (when spotted onto a sponge)
evoked a statistically significant level of feeding behaviour (Fig. 1).
Ink was highly effective at 10% full strength. Ink at 1% and 0.1% full
strength had deterrent effects on behaviour of 10e20% of crabs,
which was somewhat, but not statistically, higher than the 0% of
crabs deterred byseawater. These results argue that ink secretion is
an aversive deterrent and not a phagomimic, and that ink
contributes moretothis effect thanopaline. Thus, ink secretions are
a potential chemical defence in the field. This finding is similar to
results with other predatory species, including sea anemones and
several phylogenetically and ecologically diverse species of fishes
(Nolen et al. 1995; Sheybani et al. 2009; Nusnbaum & Derby 2010,
in press), but differs somewhat from results with California spiny
lobster, Panulirus interruptus, which can be attracted to and even
feed on ink secretion, an effect called phagomimicry (Kicklighter
et al. 2005). The effect of ink secretion on spiny lobsters can
F1F2F3 F4 F5
% Crabs rejecting
at 534 nm
at 280 nm
P3−1 P3−2 P3−3
% Crabs rejecting
% Crabs rejecting
(g/159 ml of ink)
Figure 3. Bioassay-guided fractionation of sea hare ink to identify chemical deterrents
against blue crabs. (a) Dry mass (upper graph) and bioactivity (lower graph) of frac-
tions F1eF5 from a Diaion HP20SS column. N ¼ 13,14,13,17 and 13 crabs, respectively.
(b) Peaks (P1eP4) resulting from separating fraction F4 on an HPLC C18 column using
100 mM NaClO4 in elution solvent. Upper trace shows peaks P1eP4 in the HPLC
chromatogram, and lower graph shows bioactivity of P1eP4 plus all other fractions
combined (¼other). N ¼ 15 crabs for P1eP4, N ¼ 9 crabs for ‘other’. (c) Peaks from
injection of P3 into an HPLC C18 column using isopropanol:water (5:2) isocratic
elution. Upper trace shows three peaks (P3-1, P3-2, P3-3) in the resultant chromato-
gram, and lower graph shows bioactivity of these three peaks. N ¼ 9 crabs for each
peak. Asterisks denote significant differences in the crabs’ response to the fraction and
the sea water control (one-tailed McNemar’s test: P < 0.05).
M. Kamio et al. / Animal Behaviour 80 (2010) 89e100
differ from animal to animal, and even within an animal tested
under different physiological conditions, probably because of the
complexityof the composition of ink secretionand the variability in
the physiological state of the predator. The amino acid fraction of
ink secretion can evoke phagomimicry, but its aversive compounds
can evoke avoidance and deterrence. This difference in effects
against a diversity of predatory species, even against a single
species, may explain ink secretion’s chemical complexity: each
component might work for some predators but notforothers, or for
some physiological states but not for others. Much literature on
deterrent compounds suggests that both the level of deterrent in
food and the nutritional value of food jointly determine the
response of consumers to chemical defences (e.g. Cruz-Rivera &
Hay 2003). Shrimp in our experiment may have been a particu-
larly high-quality food, and thus, opaline might be an effective
deterrent in other contexts or experimental designs.
In chemical defences, a single deterrent compound can be
effective against multiple consumer species (Cronin et al.1997; Hay
et al. 1998a; Schnitzler et al. 1998), and it can even have multiple
roles, includingantipredatory,allelopathic,antifouling and
antibacterial effects (Kubanek et al. 2002). However, a compound
can affect consumers in different ways (Paulet al.1988; Cronin et al.
1997; Hay et al. 1998a; Schnitzler et al. 1998). Thus, using a set of
compounds with a variety of effects against a diversity of enemies
will be the most effective defence in a marine ecological commu-
nity with many species. Our results in this report, together with
previous work, shows that A. californica has immense variety in its
chemical defence systems.
Since ink is not only a chemical stimulus but is also a coloured
visual stimulus, ink might be acting as a deterrent through either
the chemical or the visual modality of crabs. To reveal which
modality is more important, we performed the squirting assay with
normal crabs and with crabs with eye caps so theycould not see the
stimulus. Results were similar with both types of crabs (Fig. 2),
showing that vision is not necessary to mediate the effect of ink,
and demonstrating a central role of chemoreception. Further
evidence for this was the crabs’ behaviour towards dry shrimp
soaked in ink or active fractions of ink. Such shrimp were typically
quickly grabbed when presented to the mouthparts of crabs. Once
in the mouth, shrimp spotted with ink were manipulated and
% Crabs rejecting food
Dry mass (mg/4 ml of ink)
0 10 20
Retention time (min)
Concentration (% ink equivalents)
Concentration (% ink equivalents)
% Crabs rejecting food
at 535 nm
% Crabs rejecting food
PEB: R= H
APV: R= CH3
Fractionation by HPLC
4 ml ink
fractionation on HP20
Figure 4. Evaluation of chemical deterrents in sea hare ink. (a) Summary of fractionation of 4 ml of ink yielding four fractions, and the structure of aplysioviolin (APV) and
phycoerythrobilin (PEB). The fraction from HP20 column eluted with 40% methanol (¼40 M), and three fractions (pure APV, pure PEB, and all other fractions combined (¼OT) from
HPLC separation of a fraction) eluted with 100% methanol from HP20 column (¼100 M). (b) Relative dry mass (mg) of each fraction in 4 ml of ink. (c) Deterrence of each fraction at
equal concentration (6.25 mg/ml, which is the concentration of APV in 10% ink equivalent) (one-tailed McNemar’s test: N ¼ 10, *P < 0.05). (d) Concentrationeresponse data of
deterrence of the four fractions and ink in the behavioural bioassay based on ingestion of food-flavoured alginate pellets. Each sample at each concentration was compared to its
own control (one-tailed McNemar’s test: N ¼ 7e17, *P < 0.05). (e) Concentrationeresponse data of combinations of the four fractions. Each sample at each concentration was
compared to its own control (one-tailed McNemar’s test: N ¼ 10e11, *P < 0.05).
M. Kamio et al. / Animal Behaviour 80 (2010) 89e100
peeled, often for many minutes (Fig. 2d), unlike shrimp spotted
with sea water, which crabs quickly ate. This result suggests that
crabs are seeking portions of the ink-spotted shrimp that have little
or no ink, and supports the idea that the mouthparts contain the
chemosensors that detect the deterrent compounds in ink secre-
tion. This is consistent with what we know about the feeding
behaviour of crabs and other crustaceans: that the decision about
what is swallowed is controlled by the mouthparts (Rittschof 1992;
Tomaschko 1994; Atema & Voigt 1995; Derby et al. 2001; Garm
et al. 2005; Aggio & Derby 2008).
Aplysioviolin is a Major Deterrent in Ink against Blue Crabs
Our results show that both APV and PEB (Fig. 4a) are chemical
deterrents of the feeding behaviour of blue crabs. APV and PEB
were equally effective, on a concentration basis, in inhibiting
ingestion of food (Fig. 4c). Because the level of PEB in A. californica
ink is below PEB’s bioactive concentration, APV is the major
contributor to A. californica ink’s deterrency against blue crabs
(Fig. 4d). However, additive or synergistic effects among the
compounds may exist. Inactive fractions showed deterrence when
mixed with each other (Fig. 4e). We chose to use A. californica ink in
studies of blue crabs because A. californica is readily available and
well studied, even though it is not sympatric with blue crabs. We
evaluated the ecological relevance of sea hare ink defences,
including APV and PEB, against blue crabs by performing some
experiments on a sea hare that is sympatric with blue crabs,
A. dactylomela. We show that, as for ink of A. californica, ink of
A. dactylomela has both APV and PEB, and APV is at higher levels
than PEB (Fig. 6). Aplysia dactylomela ink had about 10 times lower
absolute concentrations of APV and PEB than A. californica ink. Ink
at 10% full strength was active in our experiment (Fig. 4d), which is
probably a relevant concentration because the mucousy consis-
tency of ink secretion keeps it from being quickly diluted in sea
water after its release. So APV in ink of A. dactylomela is likely to act
as a chemical defence as it does in A. californica’s ink. But APV in
A. dactylomela might also work in a different way from A. cal-
ifornica’s ink. For example, A. dactylomela may rely on APV less than
other defensive compounds. The significance of this comparative
study on concentration is difficult to interpret since our specimens
of both species were wild caught by commercial vendors or other
researchers and thus these species’ diets and inking histories,
which can affect levels of APV and PEB in ink, were unknown before
arriving in our laboratory.
The fact that most sea hares release purple ink (Johnson &
Willows 1999) and that APV and PEB are major contributors to
ink of at least four sea hare species (Rüdiger 1967,1994; Chapman &
Fox 1969; Appleton et al. 2001; Jongaramruong et al. 2002; this
study) indicates that our results on chemical defences of sea hares
against blue crabs are potentially ecologically relevant. APV and
PEB are probably present in most if not all sea hares and thus of
ecological significance as chemical defences against a broad arrayof
predators; however, evaluation of this idea will require measure-
ment of these compounds in the ink of other sea hare species, as
well as deterrence assays using diverse potential predators with
ecologically relevant predatoreprey combinations and more real-
Previous studies considered whether APV or PEB was used as
a chemical deterrent against predators, but none provided
conclusive demonstrations. Chapman & Fox (1969) concluded that
APV is not a chemical defence but rather is probably a waste
product because at that time there were no published studies
supporting a defensive role of APV. Carlson & Nolen (1997) reported
that chemoreceptor neurons of the crab Cancer antennarius
responded to commercially purchased phycoerythrin and molec-
ular weight fractions of ink containing purple pigments, but they
did not test either APV or PEB. Pennings et al. (1999) reported that
a fraction of ink from the sea hare Dolabella auricularia containing
the purple chromophores inhibited feeding behaviour of the
% Crabs showing food−seeking behaviour
Figure 5. Deterrence of purified aplysioviolin in squirting assay with blinded crabs.
Difference between food-seeking behaviour of crabs to shrimp juice in the presence of
APV at 16% natural concentration and in the presence of sea water (SW) control (one-
tailed McNemar’s test: N ¼ 10, *P ¼ 0.0117).
A. dactylomelaA. californica
Concentration in ink (mg/ml)
Figure 6. Concentration of aplysioviolin (APV) and phycoerythrobilin (PEB) in ink from
Aplysia californica and A. dactylomela. Values for A. californica are from a single sample
pooled from ink of 10 individual animals. Values for A. dactylomela are means ? SE
from three individual animals.
M. Kamio et al. / Animal Behaviour 80 (2010) 89e100
spotted sharpnose pufferfish Canthigaster solandri, but this fraction
was a mixture and its bioactive components were not identified.
Other biological effects of APV have been reported, but these are
not ecologically relevant as they include toxic effects against
cultured cell lines, bacteria or brine shrimp (Appleton et al. 2001;
Jongaramruong et al. 2002).
Components in ink besides APV contribute to the deterrency of
ink against blue crabs. This is most obvious from our finding that
combinations of fractions of ink lacking APV and PEB had signifi-
cant deterrency (Fig. 4e), even when individually those fractions
did not (Fig. 4d).
Crabs’ responses to these combined fractions did not appear to
be a simple addition of their response to individual components,
but we did not explore either the identity of the effective compo-
nents or the mechanisms behind their nonlinear addition of
Results testing escapin products that are produced by the
combination of escapin (in ink) and L-lysine (in opaline) showed
that all stimuli containing hydrogen peroxide were statistically
significant deterrents of feeding behaviour of blue crabs (Fig. 7b).
Other products were moderately effective deterrents (lysine, EIP:
see Fig. 7a). Thus, hydrogen peroxide is a moderate feeding deter-
rent for blue crabs. Hydrogen peroxide is also a mild feeding
deterrent for spiny lobster (Aggio & Derby 2008) and some fishes
(Nusnbaum & Derby, in press) but not for sea anemones
(Kicklighter & Derby 2006). Hydrogen peroxide is also produced by
escapin’s orthologue in Aplysia dactylomela, dactylomelin-P (Melo
et al. 2000; Yang et al. 2005).
Pigmented molecules are infrequently reported as feeding
deterrents (Wickramasinghe & Bandaranayake 2006). Notable
examples include pigmented deterrents in sponges (Amsler et al.
2001; Furrow et al. 2003) and ciliates (Miyake et al. 2001).
Handling time (s)
EEPHP SWK NH4
N= 10 1010 10 1010 10 10
N= 8010 10 10 10 101010
Bite and peel
% Crabs showing
Figure 7. Effect of products from the escapin pathway on ingestion of food by blue crabs. (a) Escapin pathway. Escapin, an L-amino acid oxidase in ink, oxidizes L-lysine(1) (K) to
convert the alpha amino group to a keto group, forming ‘escapin intermediate products’ of lysine (2e7) (EIP), hydrogen peroxide (HP) and ammonium (NH4þ), and reactions
between EIP and HP produce ‘escapin end products’ of lysine (8e9) (EEP). (b, c) Dry shrimp ingestion assay of escapin pathway products. (b) Frequency of two types of behaviours
(‘eat’ or ‘bite and peel’) shown by crabs to stimuli. Statistically significant differences from sea water (SW) controls are indicated by *P < 0.05 (one-tailed Wilcoxon signed-ranks
test: N ¼ 10). (c) Crab handling time of shrimp before ingesting shrimp (‘eat’) or completing ‘bite and peel’. Statistically significant differences from sea water (SW) controls are
indicated by *P < 0.05, **P < 0.01 (one-tailed Wilcoxon signed-ranks test: N ¼ 10). Values are medians ? interquartile ranges. All SW data (10 ? 8) are combined in this graph.
M. Kamio et al. / Animal Behaviour 80 (2010) 89e100
Escapin, the L-amino acid oxidase in sea hare ink, is yellow because
it is flavin dependent (Yang et al. 2005) and it generates feeding
deterrents against predators (Aggio & Derby 2008; Nusnbaum &
Derby, in press; this study).
Does Inking Facilitate Escape of Sea Hares from Predators in the
Inking can aid cephalopods such as squid, octopus and cuttlefish
in escaping from predators by acting as a smoke screen or decoy, or
otherwise allow a cephalopod to swim away from the predator
quickly (Wood et al., in press). In contrast, sea hares such as
A. californica and A. dactylomela cannot swim and can only move
away from a predator using relatively slow pedal locomotion
(Carefoot 1987; Johnson & Willows 1999). Nevertheless, laboratory
experiments show that inking can be an effective defence against
predatory spiny lobsters and fish (Kicklighter et al. 2005;
Nusnbaum & Derby 2010). The habitat of sea hares might help
their escape strategy: although they have a wide range of habitats
(Carefoot 1987), most habitats provide refuges that allow sea hares
to hide and avoid predators (Pennings 1990). A short hesitation of
a predator due to ink’s deterrence may contribute to escape by sea
hares, without requiring fast escape as in inking cephalopods. The
effect of inking on the survival of sea hares in the field needs to be
Sequestering a Relatively Ubiquitous Source of Deterrent
Compounds by a Flexible Specialist Herbivore
There are many reports of sequestration and conversion of food-
derived deterrent molecules to defences and de novo synthesis of
deterrent molecules for chemical defence in molluscs. There are
two biosynthetic types of molluscs: (1) species that sequester
molecules from their food and use them as deterrents, either
unmodified or after some metabolic conversion; (2) species that
produce deterrent molecules by de novo synthesis from primary
metabolites (Cimino & Ghiselin 1999, 2009; Garson 2001). Sea
hares are relatively specialized herbivores that feed mainly on red
and green algae (reviewed in Carefoot 1987) and sequester
secondary metabolites from chemically defended algae, but they
also consume a wide variety of seaweeds that do not have
secondary metabolites (reviewed in Hay & Fenical 1988). Depend-
ing on only one secondary metabolite for chemical defence is risky,
since the seaweed with this metabolite may not be available across
space and time. Using a variety of secondary metabolites from
a diversity of seaweeds is an effective way of ensuring the presence
of adequate chemical defences. Using a photosynthetic pigment
such as PE as material for chemical defence is another way of
guaranteeing a stable chemical defence because it is ubiquitous in
red algae, and, as long as sea hares feed on some species of red
algae, they can produce this chemical defence. The escapinelysine
system is another chemical defence that can be maintained on
almost any diet, since the primary metabolites for this system are
available from any diet and the bioactive molecules are produced
by sea hares themselves. Thus, these chemical defence systems of
sea hares are well supported by the sea hares’ feeding habits.
We thank Juan Aggio and Matthew Nusnbaum for assisting with
dissection and reviewing the manuscript, Cynthia Kicklighter and
Ko-Chun Ko for assisting with preparation of escapin products and
opaline extract and Barry Ache and Peter Anderson for use of
facilities at the Whitney Laboratory. Supported by National Science
Foundation grant IBN-0614685 and Naval Surface Warfare Center,
Panama City contract N61331-07-P-3004, with special thanks to
Nick Mitchell for supporting our project.
Aggio, J. F. & Derby, C. D. 2008. Hydrogen peroxide and other components in the
ink of sea hares are chemical defenses against predatory spiny lobsters acting
through non-antennular chemoreceptors. Journal of Experimental Marine
Biology and Ecology, 363, 28e34.
Amsler, C. D., McClintock, J. B. & Baker, B. J. 2001. Secondary metabolites as
mediators of trophic interactions among Antarctic marine organisms. American
Zoologist, 41, 17e26.
Appleton, D. R., Babcock, R. C. & Copp, B. R. 2001. Novel tryptophan-derived
dipeptides and bioactive metabolites from the sea hare Aplysia dactylomela.
Tetrahedron, 57, 10181e10189.
Atema, J. & Voigt, R. 1995. Behavior and sensory biology. In: Biology of the Lobster
Homarusamericanus(Ed.by J.R.Factor), pp. 313e349. New York:Academic Press.
Carefoot, T. H. 1987. Aplysia: its biology and ecology. Oceanography and Marine
Biology: an Annual Review, 25, 167e284.
Carlson, B. A. & Nolen, T. G. 1997. Defensive ink of Aplysia activates dactyl
chemoreceptors of the predatory crab Cancer. Society for Neuroscience Abstracts,
Chapman, D. J. & Fox, D. L. 1969. Bile pigment metabolism in the sea-hare Aplysia.
Journal of Experimental Marine Biology and Ecology, 4, 71e78.
Cimino, G. & Ghiselin, M. T. 1999. Chemical defense and evolutionary trends in
biosynthetic capacity among dorid nudibranchs (Mollusca: Gastropoda:
Opisthobranchia). Chemoecology, 9, 187e207.
Cimino, G. & Ghiselin, M. T. 2009. Chemical Defense and Evolution of Opisthobranch
Gastropods. San Francisco: California Academy of Sciences.
Clark, V. C., Raxworthy, C. J., Rakotomalala, V., Sierwald, P. & Fisher, B. L. 2005.
Convergent evolution of chemical defense in poison frogs and arthropod prey
between Madagascar and the Neotropics. Proceedings of the National Academy of
Sciences, U.S.A., 102, 11617e11622.
Coelho, L., Prince, J. & Nolen, T. G.1998. Processing of defensive pigment in Aplysia
phycoerythrin by the digestive gland. Journal of Experimental Biology, 201,
Cronin, T. W. & Forward, R. B. Jr. 1988. The visual pigments of crabs. I. Spectral
characteristics. Journal of Comparative Physiology A, 162, 463e478.
Cronin, G., Paul, V. J., Hay, M. E. & Fenical, W. 1997. Are tropical herbivores
more resistant than temperate herbivores to seaweed chemical defenses?
Diterpenoid metabolites from Dictyota acutiloba as feeding deterrents for
tropical versus temperate fishes and urchins. Journal of Chemical Ecology, 23,
Cruz-Rivera, E. & Hay, M. E. 2003. Prey nutritional quality interacts with chemical
defenses to affect consumer feeding and fitness. Ecological Monographs, 73,
Derby, C. D. 2007. Escape by inking and secreting: marine molluscs avoid predators
through a rich array of chemicals and mechanisms. Biological Bulletin, 213,
Derby, C. D. & Sorensen, P. W. 2008. Neural processing, perception, and behavioral
responses to natural chemical stimuli by fish and crustaceans. Journal of
Chemical Ecology, 34, 898e914.
Derby, C. D., Steullet, P. & Horner, A. J. 2001. The sensory basis to feeding behavior
in the Caribbean spiny lobster Panulirus argus. Marine & Freshwater Research,
DiMatteo, T. 1981. The inking behavior of Aplysia dactylomela (Gastropoda: Opis-
thobranchia): evidence for distastefulness. Marine Behaviour and Physiology, 7,
DiMatteo, T. 1982. The ink of Aplysia dactylomela (Rang, 1828) (Gastropoda: Opis-
thobranchia) and its role as a defensive mechanism. Journal of Experimental
Marine Biology and Ecology, 57, 169e180.
Eisner, T. & Aneshansley, D. J. 1999. Spray aiming in the bombardier beetle:
photographic evidence. Proceedings of the National Academy of Sciences, U.S.A.,
Furrow, F. B., Amsler, C. D., McClintock, J. B. & Baker, B. J. 2003. Surface
sequestration of chemical feeding deterrents in the Antarctic sponge Latrun-
culia apicalis as an optimal defense against sea star spongivory. Marine Biology,
Garm, A., Shabani, S., Høeg, J. & Derby, C. D. 2005. Chemosensory neurons in the
mouthparts of the spiny lobsters Panulirus argus and Panulirus interruptus
(Crustacea: Decapoda). Journal of Experimental Marine Biology and Ecology, 314,
Garson, M. J. 2001. Ecological perspectives on marine natural product biosynthesis.
In: Marine Chemical Ecology (Ed. by J. B. McClintock & B. J. Baker), pp. 71e114.
Boca Raton, Florida: CRC Press.
Ginsburg, D. W. & Paul, V. J. 2001. Chemical defenses in the sea hare Aplysia
parvula: importance of diet and sequestration of algal secondary metabolites.
Marine Ecology Progress Series, 215, 261e274.
Hadacek, F. 2002. Secondary metabolites as plant traits: current assessment and
future perspectives. Critical Reviews in Plant Sciences, 21, 273e322.
Hay, M. E. & Fenical, W. 1988. Marine planteherbivore interactions: the ecology of
chemical defense. Annual Review of Ecology and Systematics, 19, 111e145.
M. Kamio et al. / Animal Behaviour 80 (2010) 89e100
Hay, M. E., Piel, J., Boland, W. & Schnitzler, I.1998a. Seaweed sex pheromones and
their degradation products frequently suppress amphipod feeding but rarely
suppress sea urchin feeding. Chemoecology, 8, 91e98.
Hay, M. E., Stachowicz, J. J., Cruz-Rivera, E., Bullard, S., Deal, M. S. & Lindquist, N.
1998b. Bioassays with marine and freshwater macroorganisms. In: Methods in
Chemical Ecology. Vol. 2. Bioassay Methods (Ed. by K. F. Haynes & J. G. Millar), pp.
39e141. New York: Chapman & Hall.
Hines, A. H. 2007. Ecology of juvenile and adult blue crabs. In: The Blue Crab Cal-
linectes sapidus (Ed. by V. S. Kennedy & L. E. Cronin), pp. 565e654. College Park:
Maryland Sea Grant College.
Johnson, P. M., Kicklighter, C. E., Schmidt, M., Kamio, M., Yang, H., Elkin, D.,
Michel, W. C., Tai, P. C. & Derby, C. D. 2006. Packaging of chemicals in the
defensive secretory glands of the sea hare Aplysia californica. Journal of Exper-
imental Biology, 209, 78e88.
Johnson, P. M. & Willows, A. O. D. 1999. Defense in sea hares (Gastropoda, Opis-
thobranchia, Anaspidea): multiple layers of protection from egg to adult. Marine
and Freshwater Behaviour and Physiology, 32, 147e180.
Jongaramruong, J., Blackman, A. J., Skelton, B. W. & White, A. H. 2002.
Chemical relationships between the sea hare Aplysia parvula and the red
seaweed Laurencia filiformis from Tasmania. Australian Journal of Chemistry, 55,
Kamio, M., Ko, K.-C., Zheng, S., Wang, B., Collins, S. L., Gadda, G., Tai, P. C. &
Derby, C. D. 2009. The chemistry of escapin: identification and quantification of
the components in the complex mixture generated by an L-amino acid oxidase
in the defensive secretion of the sea snail Aplysia californica. Chemistry e
a European Journal, 15, 1597e1603.
Kamiya, H., Sakai, R. & Jimbo, M. 2006. Bioactive molecules from sea hares. In:
Molluscs: from Chemo-ecological Study to Biotechnological Application. Progress in
Molecular Biology. Marine Molecular Biotechnology (Ed. by G. Cimino &
M. Gavagnin), pp. 215e239. Berlin: Springer-Verlag.
Kicklighter, C. E. & Derby, C. D. 2006. Multiple components in ink of the sea hare
Aplysia californica are aversive to the sea anemone Anthopleura sola. Journal of
Experimental Marine Biology and Ecology, 334, 256e268.
Kicklighter, C. E., Shabani, S., Johnson, P. M. & Derby, C. D. 2005. Sea hares use
novel antipredatory chemical defenses. Current Biology, 15, 549e554.
Kubanek, J., Whalen, K. E., Engel, S., Kelly, S. R., Henkel, T. P., Fenical, W. &
Pawlik, J. R. 2002. Multiple defensive roles for triterpene glycosides from two
Caribbean sponges. Oecologia, 131, 125e136.
McClintock, J. B. & Baker, B. J. (Eds). 2001. Marine Chemical Ecology. Boca Raton,
Florida: CRC Press.
Melo,V.M. M.,Duarte,A.B.G., Carvalho,A.F.F.U., Siebra,E.A.&Vasconcelos, I. M.
2000. Purification of a novel antibacterial and haemagglutinating protein from
the purple gland of the sea hare, Aplysia dactylomela Rang, 1828. Toxicon, 38,
Miyake, A., Harumoto, T. & Iio, H. 2001. Defence function of pigment granules in
Stentor coeruleus. European Journal of Protistology, 37, 77e88.
Nolen, T. G., Johnson, P. M., Kicklighter, C. E. & Capo, T. 1995. Ink secretion by the
marine snail Aplysia californica enhances its ability to escape from a natural
predator. Journal of Comparative Physiology A, 176, 239e254.
Nusnbaum, M. & Derby, C. D. In press. Effects of sea hare ink secretion and its
escapin-generated components on a variety of predatory fishes. Biological
Nusnbaum, M. & Derby, C. D. 2010. Ink secretion protects sea hares by acting on
the olfactory and non-olfactory chemical senses of a predatory fish. Animal
Behaviour, 79, 1067e1076.
Paul, V. J. & Pennings, S. C. 1991. Diet derived chemical defenses in the sea hare
Stylocheilus longicauda (Quoy and Gimard 1824). Journal of Experimental Marine
Biology and Ecology, 151, 227e243.
Paul, V. J. & Ritson-Williams, R. 2008. Marine chemical ecology. Natural Product
Reports, 25, 662e695.
Paul, V. J., Hay, M. E., Duffy, J. E., Fenical, W. & Gustafson, K. 1988. Chemical
defense in the seaweed Ochtodes secundiramea. Effects of its monoterpenoid
components upon diverse coral-reef herbivores. Journal of Experimental Marine
Biology and Ecology, 114, 249e260.
Pearson, W. H. & Olla, B. L. 1977. Chemoreception in the blue crab, Callinectes
sapidus. Biological Bulletin, 153, 346e354.
Pennings, S. C.1990. Predatoreprey interactions in opisthobranch gastropods: effects
Pennings, S. C., Paul, V. J., Dunbar, D. C., Hamann, M. T., Lumbang, W. A.,
Novack, B. & Jacobs, R. S. 1999. Unpalatable compounds in the marine
gastropod Dolabella auricularia: distribution and effect of diet. Journal of
Chemical Ecology, 25, 735e755.
Prince, J. S. & Johnson, P. M. 2006. Ultrastructural comparison of Aplysia and
Dolabrifera ink glands suggests cellular sites of anti-predator protein production
and algal pigment processing. Journal of Molluscan Studies, 72, 349e357.
Prince, J. S., Nolen, T. G. & Coelho, L. 1998. Defensive ink pigment processing and
secretion in Aplysia californica: concentration and storage of phycoerythrobilin
in the ink gland. Journal of Experimental Biology, 201, 1595e1613.
Rittschof, D.1992. Chemosensation in the daily life of crabs. American Zoologist, 32,
Rüdiger, W.1967. Uber die Abwehrfarbstoffe von Aplysia-Arten, II. Die Struktur von
Aplysioviolin. Hoppe-Seyler's Zeitschrift für physiologische Chemie, 348, 1554.
Rüdiger, W. 1994. Phycobiliproteins and phycobilins. In: Progress in Phycological
Research. Vol. 10 (Ed. by F. E. Round & D. J. Chapman), pp. 97e135. Bristol:
Schnitzler, I., Boland, W. & Hay, M. E. 1998. Organic sulfur compounds from Dic-
tyopteris spp. deter feeding by an herbivorous amphipod (Ampithoe longimana)
but not by an herbivorous sea urchin (Arbacia punctulata). Journal of Chemical
Ecology, 24, 1715e1732.
Sheybani, A., Nusnbaum, M., Caprio, J. & Derby, C. D. 2009. Responses of the sea
catfish, Ariopsis felis, to chemical defenses from the sea hare, Aplysia californica.
Journal of Experimental Marine Biology and Ecology, 368, 153e160.
Shiomi, K., Yokota, H., Nagashima, Y. & Ishida, M. 2001. Primary and secondary
structures of grammistins, peptide toxins isolated from the skin secretion of the
soapfish Pogonoperca punctata. Fisheries Science, 67, 163e169.
Thoms, C. & Schupp, P. J. 2008. Activated chemical defense in marine sponges:
a case study on Aplysinella rhax. Journal of Chemical Ecology, 34, 1242e1252.
Tomaschko, K. H. 1994. Ecdysteroids from Pycnogonum litorale (Arthropoda, Pan-
topoda) act as chemical defense against Carcinus maenas (Crustacea, Decapoda).
Journal of Chemical Ecology, 20, 1445e1455.
Van Alstyne, K. L. & Houser, L. T. 2003. Dimethylsulfide release during macro-
invertebrate grazing and its role as an activated chemical defense. Marine
Ecology Progress Series, 250, 175e181.
Walling, L. L. 2009. Adaptive defense responses to pathogens and insects. In: Plant
Innate Immunity. Vol. 51: Advances in Botanical Research (Ed. by L. C. van Loon),
pp. 551e612. London: Academic PresseElsevier Science.
Walters, E. T. & Erickson, M. T. 1986. Directional control and the functional
organization of defensive responses in Aplysia. Journal of Comparative Physiology
A, 159, 339e351.
Weissburg, M. J. & Zimmer-Faust, R. K.1994. Odor plumes and how blue crabs use
them in finding prey. Journal of Experimental Biology, 197, 349e375.
Wickramasinghe, M. & Bandaranayake, M. 2006. The nature and role of pigments
of marine invertebrates. Natural Product Reports, 23, 223e255.
Williams, J. R. & Gong, H. 2007. Biological activities and syntheses of steroidal
saponins: the shark-repelling pavoninins. Lipids, 42, 77e86.
Wink, M. 2008. Plant secondary metabolism: diversity, function and its evolution.
Natural Product Communications, 3, 1205e1216.
against herbivores and pathogens. Current Opinion in Plant Biology, 5, 300e307.
Wood, W. F. 1999. The history of skunk defensive secretion research. Chemical
Educator, 4, 44e50.
Wood, J. B., Pennoyer, K. E. & Derby, C. D. 2008. Ink is a conspecific alarm cue in
the Caribbean reef squid, Sepioteuthis sepioidea. Journal of Experimental Marine
Biology and Ecology, 367, 11e16.
Wood, J. B., Maynard, A., Lawlor, A., Sawyer, E. K., Simmons, D., Pennoyer, K. E. &
Derby, C. D. In press. Caribbean reef squid, Sepioteuthis sepioidea, use ink as
a defense against predatory French grunts, Haemulon flavolineatum. Journal of
Experimental Marine Biology and Ecology.
Yang, H., Johnson, P. M., Ko, K.-C., Kamio, M., Germann, M. W., Derby, C. D. &
Tai, P. C. 2005. Cloning, characterization and expression of escapin, a broadly
antimicrobial FAD-containing L-amino acid oxidase from ink of the sea hare
Aplysia californica. Journal of Experimental Biology, 208, 3609e3622.
M. Kamio et al. / Animal Behaviour 80 (2010) 89e100