Journal of Experimental Marine Biology and Ecology,
234 (1999) 185–197
L
A test of novel function(s) for the ink of sea hares
Thomas H. Carefoot a , *, Steven C. Pennings b , Jean Paul Danko a
a
Department of Zoology, University of British Columbia, 6270 University Blvd, Vancouver, BC V6 T 1 Z4,
Canada
b
Marine Institute, University of Georgia, Sapelo Island, GA 31327, USA
Received 12 March 1998; received in revised form 20 August 1998; accepted 21 August 1998
Abstract
Most sea hares (Opisthobranchia: Anaspidea) release a purple ink when physically disturbed.
The ink has been hypothesized to function to excrete unwanted byproducts of metabolism, as a
smoke screen, as an anti-feedant, and as a warning signal. We tested two additional potential
functions: that ink is a metabolic depressant and / or a noxious or adversive sensory stimulus. When
exposed to realistic concentrations of ink from Aplysia dactylomela (Rang), none of five
invertebrate species (including A. dactylomela) or two fish species significantly altered their
oxygen uptake, and neither of two crab species significantly altered their heart and / or scaphognathite beat rates, suggesting that ink does not function as a metabolic depressant. In contrast,
although A. dactylomela did not display strong behavioural responses to ink, behaviour of seven
other invertebrates and both fish species was strongly affected by ink, supporting our hypothesis
that the ink functions as an irritant. Observed behavioural changes included bristle erection by
fireworms, increased mucus production by an opisthobranch, reduced feeding behaviour, increased
grooming behaviour, and temporary pauses in heart and scaphognathite beating by crabs, reduced
and increased activity by cryptic and exposed sea urchin species, respectively, and rapid
swimming by fish. Similar behavioural changes by potential predators would likely lead to reduced
predation rates on Aplysia spp. in the field. Our conclusion that ink functions as a sensory irritant
is not incompatible with other hypotheses for the function of ink.  1999 Elsevier Science B.V.
All rights reserved.
Keywords: Sea hare; Aplysia; Ink; Defensive function; Opisthobranchia
1. Introduction
Most sea hares (specifically, Aplysia spp., but including closely related Dolabella spp.
*Corresponding author. Tel.: 11-604-8224357; e-mail: [email protected]
0022-0981 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved.
PII: S0022-0981( 98 )00153-1
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T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 – 197
and Stylocheilus spp.) (Opisthobranchia:Anaspidea) produce a purple-coloured ink from
special glands in the lower surface of the mantle cavity. The primary organic constituent
of the ink is aplysioviolin, a violet-colored ester derived from phycoerythrobilin, a
pigment of red seaweeds eaten as food (Chapman and Fox, 1967). If these seaweeds are
omitted from the diet, an animal becomes facultatively de-inked. On its release, the ink
consists mostly of water and other volatile substances with less than 2% dry mass
organic substances and 5% minerals (Flury, 1915). Ink volume in the glands has never
been measured, but even in a large animal could comprise no more than a few milliliters
(personal observation). Release is accompanied by varying degrees of mantle contractions and parapodial flappings which, depending upon their magnitude and the amount of
mucus released with the ink, will cause the ink to hang in a heavy purple cloud in the
water near the animal or be dispersed more widely and quickly.
Release of ink is a high-threshold, all-or-none response (Carew and Kandel, 1977;
Shapiro et al., 1979; Byrne, 1981). In the laboratory it can often, but not always, be
induced by rough handling, pin pricks, mild electric shocks, separation of copulating
individuals, or the like. In the field, observation of ink release in the absence of human
stimulation is extremely rare (Kupfermann and Carew, 1974; our personal observation).
Only Willan (1979) has reported seeing ink released in response to actions of another
animal in the field, in this case an Aplysia dactylomela apparently under attack from the
starfish Coscinasterias calamaria.
Indeed, predators of Aplysia species are essentially unknown (for review see Carefoot,
1987; Pennings, 1990a). Apart from ink as a possible first line of defense, sea hares
possess a toxic opaline-gland secretion (Flury, 1915; Ando, 1952) and a broad spectrum
of algal-derived toxins in the skin and digestive gland (for review see Carefoot, 1987).
Aplysia dactylomela alone sequesters some 20 different secondary metabolites from its
algal diet, any or all of which could be defensive in function. However, despite the depth
and diversity of this chemical repertoire, unequivocal demonstration of a defensive
function for any component is mostly lacking (Beeman, 1961; Ambrose et al., 1979;
DiMatteo, 1981, 1982; Pennings, 1990b; Paul and Pennings, 1991; Pennings and Paul,
1993; Pennings, 1994; Nolen et al., 1995).
Several hypotheses have been proposed for the function of ink in Aplysia and related
sea hares: (1) it acts as a method to rid the animal of unwanted bile pigments consumed
in its diet (Chapman and Fox, 1967), (2) it acts as a ‘smoke-screen’ on release, thus
shielding the sea hare from visual predators (Eales, 1921; Halstead, 1965; Hyman, 1967;
Carew and Kandel, 1977), (3) it is distasteful, causing the sea hare to be unpalatable and
thus acting as an ‘anti-feedant’ (Beeman, 1961; DiMatteo, 1981, 1982; Pennings, 1994;
Nolen et al., 1995), (4) it functions as a warning to would-be predators of the sea hare’s
other toxic properties (Ambrose et al., 1979), and (5) it acts in some manner as an alarm
signal to conspecifics (Fiorito and Gherardi, 1990; Stopfer et al., 1993; this last idea is
consistent with the notion that cephalopod ink may function as an intraspecific alarm
substance: Gilly and Lucero, 1992, and others). However, based on the observation of
Willan (1979) that locomotory movement of the starfish Coscinsterias calamaria was
retarded by contact with sea-hare ink, additional hypotheses of metabolic depressanteffect or chemosensory desensitization could be added to the list. This last idea is
reminiscent of the observation of MacGinitie and MacGinitie (1968) that the ‘real effect’
of octopus ink is to anaesthetize the chemosensory abilities of fish predators.
T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 – 197
187
In this paper we test the hypothesis that ink has a metabolic inhibitory effect on other
organisms. We have long noticed that Aplysia spp. exposed to their own ink appear
largely unaffected if the duration of exposure is relatively short and ink concentration
not excessive, while other animals such as crabs and fish can be seriously and often
fatally affected by the same exposure. In addition, we test another hypothesis that the ink
functions as a chemosensory irritant to ward off attacks by potential predators. This
hypothesis is based on our observations that crabs often attempt to groom themselves if
they contact ink and mucus from Aplysia spp. (see also DiMatteo, 1982). We
hypothesized that ink might act to protect the sea hares at a distance (i.e. without direct
contact and taste) by irritating potential predators enough that they would leave the
vicinity of the sea hare. To test these hypotheses, we measured the effects of different
concentrations of sea-hare ink on the behaviour and metabolism of Aplysia dactylomela
(Rang) and on a diversity of sympatric taxa which might be naturally exposed to the ink.
2. Methods
Aplysia dactylomela is a large (to ca. 500 g) circumtropical sea hare. It is active at
night, feeding primarily on red seaweeds. The behaviour and ecology of A. dactylomela
are described in Carefoot (1989).
Aplysia dactylomela of 30–500 g live mass were collected from the shallow backreef
area near the Discovery Bay Marine Laboratory, Jamaica (188309N, 778209W) and kept
in a flow-through seawater system with an abundant supply of red algae as food.
Additionally, several species of other invertebrates and fish were collected from the
Aplysia habitat and kept in the laboratory seawater tanks (the predatory fireworm
Hermodice carunculata Hartman, green clinging crab Mithrax sculptus Lamarck,
swimming crab Portunus sebae Latrielle, sea urchins Echinometra lucunter Linnaeus
and Lytechinus variegatus Lamarck, opisthobranch Tridachia crispata Younge &
Nicholas, predatory puffer Diodon holocanthus Linnaeus, and the predatory goby
Gnatholepis thompsoni Jordan. Our goal was not to identify particular Aplysia predators
as such, since these are not well known, but rather to assess the effects of ink on a wide
taxonomic range of animals. Consequently, test species were chosen based on abundance, taxonomic diversity, and ease of collection and maintenance in the laboratory.
However, we note that the majority of the taxa selected (fish, crabs, polychaetes) include
species that are predatory upon soft-bodied invertebrates, and thus represent taxa that
include potential predators of Aplysia. The smaller Aplysia (30–60 g live mass) were
used in the respirometry and behaviour experiments, while the larger ones were
maintained as ink donors.
2.1. Collection of ink for testing
We collected ink by carefully lifting individual Aplysia out of the water and allowing
excess water to drain, then gently massaging the surface of the mantle in the vicinity of
the ink gland, shell, and gill. This procedure stimulated animals to release ink, which
was collected in a small beaker. Several animals were usually de-inked at one time to
provide sufficient ink for a number of experiments. The ink was kept at 58C between
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experiments to minimize any potential ‘aging’ effects (ink was typically used in
experiments within an hour of collection, and was never kept longer than 6 h). After an
animal was de-inked it was allowed to rest and feed for several days before ink was
collected from it again. Concentrations of ink solutions used in the tests were read as
absorbencies at 560 nm in a spectrophotometer. These values were then related to a
previously calculated regression equation of absorbency over dry mass concentration of
ink determined from 14 individual sea hares of 40–480 g live mass collected fresh, then
de-inked within 1–2 days. This allowed ink concentration to be expressed as mg dry
mass (ml seawater)21 (see Section 3).
2.2. Effect of ink on behaviour
We observed the effect of diluted Aplysia dactylomela ink on the behaviour of the
invertebrates listed earlier, as well as on the sea hare itself. Animals were tested within
48 h of collection and then released.
Individual test organisms were placed in a small volume of seawater in beakers or
fingerbowls and allowed 5 min to acclimate. Ink was then added by pipette to the water
containing the experimental animals, and seawater was added as a disturbance control to
paired control animals. Experimental and control animals were paired by size and were
tested simultaneously. Behavioural observations were made 1 and 5 min after ink
addition. The absorbency of the ink–water solution in the bowls was measured
immediately following the behavioural assays. Ink dosages were varied, but ranged
around a mean absorbency which mimicked the concentration of ink that could be
produced by a standard-sized 300-g live-mass animal if it were to ink fully into 3 l of
seawater (our estimate of effective range; see Section 3). Balanced with this was the
need in the behavioural experiments to test the ink at a concentration dilute enough so
that we could clearly observe what an animal was doing.
We made different observations on each species of test animal depending on its
specific behaviour. Each animal was scanned within each of the two observational
periods and each activity tallied, then averaged. Behaviours were, for Hermodice, rate of
locomotion (scored 0–4), bristle extension (retracted versus extended (scored 0 or 1)),
and body extension (contracted versus normal posture (scored 0 or 1)); for Mithrax,
moving (active movement of the entire animal), grooming (rubbing or scraping the claws
across or picking at the eyes, mouthparts, legs, or carapace), and feeding (repeatedly
sweeping the claw tips across the substratum and then bringing them to the mouthparts),
all scored 1–10; for Echinometra and Lytechinus, activity of spines, tube feet and podia,
and locomotion, all scored 1–4; and for Tridachia and Aplysia, qualitative observations
on parapodial movement and general activity. Additionally, we squirted 100 ml of pure
ink into the inhalent siphons of five large resting adult Aplysia and the same volume of
seawater into the siphons of five control animals.
For all quantitative observations, scores of paired experimental and control animals
were compared with paired t-tests or, in the case of binary data, with sign tests.
2.3. Oxygen consumption, heart rate, and scaphognathite rate
Oxygen consumption (VO 2 ) before and after exposure to ink was measured in the same
T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 – 197
189
species used in the behaviour experiments with the exception of the sea urchin
Lytechinus variegatus, for which insufficient numbers could be found. In addition, two
potential fish predators on Aplysia, the puffer Diodon holocanthus (Linnaeus) (10-g
live-mass juveniles only) and the goby Gnatholepis thompsoni Jordan were included in
the oxygen-uptake studies. Closed-system respirometers of different volumes (28, 84,
712, and 1265 ml) were used depending on the size of the animal being tested. Within
the respirometer, a plastic mesh floor separated the animal chamber from a rotating
stir-rod, the action of which ensured complete mixing of the water contained within the
respirometer. A Clark-type O 2 electrode (Yellow Springs Inc.) extended into the animal
chamber through a rubber bung inserted in the roof or side of the chamber, and signals
from this led into a DataQ Instruments, data-acquisition and analysis software system.
Oxygen uptake was measured as mg O 2 ?g live animal 21 at 288C and 32‰. To eliminate
size as a factor in the statistical analyses, all measured VO 2 values for a particular species
were converted to equivalent rates for a ‘standard animal’ of average adult mass of that
species using
VO 2 (x mass (g)) 5 (x / exp. mass (g))b ?VO 2 (exp.),
where x represents 30 for Aplysia and Echinometra, 10 for Diodon, 5 for Hermodice, 2.5
for Tridachia, 2 for Gnatholepis, and 1.25 for Mithrax.; and b is the slope of regression
of VO 2 against body mass for each test species). A typical run consisted of selecting an
animal and placing it in an appropriate-sized respirometer. Fresh seawater was run
through the respirometer until the animal became quiescent, after which baseline VO 2 was
measured over a time sufficient to give a significant negative slope of oxygen
concentration (PO 2 ) over time (usually 15–20 min). Following this, a known amount of
freshly collected ink was injected into the respirometer with an hypodermic syringe, and
VO 2 recorded for a further 15–20 min. Actual duration of runs varied with the type and
size of animals, but at no time was PO 2 allowed to drop below 70% saturation level.
Behaviour of control animals under these conditions appeared normal. Each species was
treated with a dosage-series of ink concentrations, using a different animal for each dose.
Actual concentration of ink used in each test was determined from spectrophotometer
readings taken at the conclusion of the test. As in the behaviour experiments, we
included a control series of oxygen-uptake measurements using similar-sized animals but
with equal volumes of seawater substituted for the ink injection. An additional control
series was conducted using ink only in the respirometer to determine if any oxidative
processes were occurring with the ink itself.
Heart and scaphognathite rates were measured only for the crabs Portunus sebae and
Mithrax sculptus. Specimens were implanted with electrodes on either side of the heart
or on either side of one of the branchial regions depending on what was being recorded.
The electrodes consisted of two [18 hypodermic syringe needles soldered to wires
leading to an impedance pneumograph device (IPV, CA) which, in turn, conducted
signals to the data-acquisition and analysis software system described above. The
implanted specimen was then strapped to a plastic ruler with plastic twist-ties and
immersed in seawater (288C) in a 1-l beaker. The seawater was mixed by a rotating
magnetic stir-bar. A typical experiment consisted of implanting and immersing a crab,
allowing it to rest for 5 min in the beaker during which the beat rate was recorded,
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T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 – 197
injecting a known amount of seawater into the beaker water, recording for a further 5
min, injecting the same volume of ink, then recording for a further 7 min. At 1–2-min
intervals over this period we analysed for beat-frequency over a 30-s portion of the
recording. Additionally, we monitored each record for information on incidence and
duration of extended stoppages of beat, something which often occurred just after the ink
was added.
‘Before’ and ‘after’ oxygen consumption data for each water or ink treatment were
analysed with repeated-measures ANOVA (R-MANOVA) coupled with Neuman–Keuls
multiple-comparisons tests (N-K tests). Data for beat-frequency of heart and scaphognathite were similarly analysed with R-MANOVA using time as the main factor.
Significance of regressions was tested using t-test analyses as outlined in Zar (1974).
3. Results
3.1. Collection of ink for testing
Ink volume was significantly related to sea-hare size (log Vol5 22.0411 log Live
mass; n514, r 2 50.69, t55.2, P50.0002). The slope, b, of 1.0 was the expected
isometric scaling of ink volume on live mass. The ink was 5.9%60.2 S.D. dry mass
(n514), similar to the value of 5.2% reported by Flury (1915) for Aplysia spp. An
average-sized animal of 300 g live mass produced 3.2 ml ink, a volume which, if
released all at once, could affect 3 l of seawater at a concentration of 60 mg ml 21 or an
absorbency of 0.06. On this basis we structured our dosage curves around mean
absorbencies of between 0.03 and 0.07 for the behaviour studies and between 0.06 and
0.18 for the physiological studies, depending on species.
3.2. Effect of ink on behaviour
Ink concentration within the range we studied had no significant effect on behaviour
for any of the animals tested (t values all ,1.2, P values all 0.12). Consequently, all
concentrations were pooled and data expressed simply as a comparison between treated
(ink) and control (seawater) animals.
Ink strongly affected Hermodice’ s behaviour (Fig. 1a). In the presence of ink the
worms crawled significantly less, and were significantly more likely to expose their
bristles and adopt a contracted body posture. Mithrax’ s locomotion was unaffected (Fig.
1b), but they groomed significantly more and exhibited significantly fewer feeding
movements in the presence of ink. Echinometra (Fig. 1c) showed significantly reduced
tube-foot activity in the presence of ink, but spine and locomotory activity were
unaffected. In comparison, all behaviours monitored for Lytechinus (Fig. 1d), including
activity of spines, tube-feet and pedicellariae, and overall locomotion were significantly
higher in the presence of ink.
Qualitative observations on the opisthobranch Tridachia crispata showed that at low
concentrations (,0.06 mg ml 21 ), ink stimulated initial fluttering of the parapodia that
quickly subsided, while at high concentrations (.0.15 mg ml 21 ) the animals produced
T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 – 197
191
Fig. 1. Effect of ink on behaviour of invertebrates: (a) Hermodice carunculata (BRISTLES, bristles retracted
(scored 0) or extended (scored 1); BODY EXT, body extension (contracted scored 0, normal scored 1)); (b)
Mithrax sculptus; (c) Echinometra lucunter; (d) Lytechinus variegatus (pedicell5pedicellaria). n516–27 pairs
for all species; data indicate means6S.E.; data were analysed with paired t-tests or, for binary data, with sign
tests.
copious mucus. Aplysia was not noticeably affected by its own ink. Pure ink squirted
into the inhalent respiratory streams of five resting Aplysia elicited the same response in
all: one to two large ventilatory exhalations. An equal volume of seawater squirted into
control animals elicited no response. Clearly, on sensing ink in its mantle cavity an
Aplysia responds as it does to its own ink, by immediately expelling it.
3.3. Effect of ink on oxygen consumption, heart rate, and scaphognathite rate
No significant relationship was found for change in oxygen consumption versus ink
dose within the range tested for any species (t values all ,1.13, P values all .0.27).
Consequently, as in the behaviour section, concentrations were pooled and data analysed
T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 – 197
192
as ink-treated versus control. Ink alone in the respirometer showed less than 2%
reduction in PO 2 over the normal duration of an experiment (about 30 min). A common
feature of all respirometry runs was that VO 2 decreased from the ‘before’ to ‘after’ parts
of each run, regardless of whether ink or seawater was added to the respirometer (Table
1). This was likely due to the tendency of an animal to become more quiescent in the
respirometer over time, and should not have interfered with the ability of the statistical
analyses to identify effects of ink on an animal’s metabolic rate. In fact, before / after
depression in VO 2 was significant only for the goby, Gnatholepis (Table 1; F 59.92,
P50.005, R-MANOVA), but neither treatment (ink vs water; F 50.37, P50.55), nor the
interaction of treatment and time (before vs after) for this species was significant
(F 50.09, P50.77). For all other species, neither treatment (F values all ,1.31, p
values all .0.26), time (F values all ,3.83, P values .0.06), nor the interaction of
treatment and time (F values all ,2.47, P values .0.12) were significant. We conclude
from this part of the study that sea-hare ink does not significantly affect oxygen
consumption of these particular invertebrates and vertebrates over the time-periods and
concentrations chosen.
Neither the puffer nor goby showed significant effects of ink on VO 2 over a
concentration range of 0.02–0.20 mg ml 21 . However, at the high end of the range, two
puffers and one goby died a few hours after the respirometry experiment had finished.
Both puffers were fully inflated prior to death, suggestive of stress.
Heart rates (Mithrax and Portunus) and scaphognathite rates (Portunus only) showed
small but non-significant depressions after addition of ink (Figs. 2 and 3, P.0.05 for all,
R-MANOVA). As was found in the experiments on oxygen uptake and behaviour, ink
concentration had no significant effect on heart rate in either species, nor on scaphognathite rate in Portunus (t,0.8, P.0.2 for all). In Portunus, but not in Mithrax, the
heart temporarily stopped beating soon after the ink was added. This occurred in 14 of
17 animals, commenced on average 30 s after ink addition, and lasted for 14 s.
Scaphognathites in Portunus also exhibited beat cessation. This occurred in four of six
Table 1
Oxygen consumption (VO 2 ) of various invertebrates and vertebrates treated with sea-hare ink or seawater
(control)
Genus
Standard
N
size
(live g)
Hermodice
5
Mithrax
1.25
Echinometra 30
Tridachia
2.5
Aplysia
30
Diodon
10
Gnatholepis
2
21
26
22
21
24
6
7
Ink
N
VO 2 before
VO 2 after
% Diff.
0.6660.36
0.4360.17
0.3260.18
0.3760.31
3.461.2
6.962.7
0.9060.61
0.5260.37
0.3860.18
0.2760.24
0.2960.13
2.761.2
5.562.6
0.4760.27
221
212
216
222
220
220
248*
22
23
25
15
23
6
7
Seawater
VO 2 before
VO 2 after
% Diff.
0.8060.52
0.4960.21
0.2960.17
0.3160.16
3.461.3
7.864.1
1.0760.30
0.5860.21
0.3560.17
0.2660.13
0.1760.09
3.161.1
5.962.7
0.6660.17
228
229
210
245
212
224
238*
Values for VO 2 are mean mg O 2 ?indiv 21 ?h 21 (6S.E.) for the idicated number of individuals of the ‘standard
size’ shown (see text for details). The asterisks indicate significant difference between ‘before’ and ‘after’ rates
within a given treatment F 59.92, P50.005, R-MANOVA).
T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 – 197
193
Fig. 2. Effect of ink on heart rates of decapod crustaceans, Portunus sebae and Mithrax sculptus. Each point
for Portunus is the mean6S.E. for n517 and for Mithrax for n57.
specimens, commenced 20 s after addition of ink and lasted for an average of 10 s. Heart
and scaphognathite cessation was not noted following addition of seawater.
4. Discussion
Our results do not support the hypothesis that sea-hare ink is a metabolic depressant,
Fig. 3. Effect of sea-hare ink on scaphognathite-beat frequencies in Portunus sebae. Each point is the
mean6S.E. for n56.
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T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 – 197
but do support the hypothesis that it is a sensory irritant. None of the species tested
displayed a significant reduction in oxygen uptake in the presence of ink. Some
behaviours were altered in the presence of ink and, save for the fact that the affected
behaviours were ones that were either short-lived (swimming), not likely energy-costly
(bristle erection), or resulting by their action in a reduction of gas-exchange surface area
(tubefoot withdrawal), we might have expected oxygen consumption to have actually
increased. In this regard, certain responses could also have offset one another in terms of
metabolic cost. Examples of this are bristle erection in Hermodice in the presence of ink
which was accompanied by decreased locomotion, and extra grooming in Mithrax which
was accompanied by reduced feeding movements. Unfortunately, the species that
showed the most consistent and potentially most energetically costly responses to ink,
Lytechinus variegatus, was also the one for which we could find too few specimens on
which to do a complete respirometry series. The responses of the sea urchins were
generally consistent with their respective habits of life. Echinometra lucunter is
sedentary and inhabits crevices and depressions in live coral or coral rocks. Its response
to ink was a pulling in of its tube-feet and no change in locomotion. In contrast, L.
variegatus is a free-ranging species that lives openly in sea-grass beds. Its response to
ink was a mobilization of its full defensive and escape repertoire, including spine
movement, pedicellariae activity, and increased locomotion.
These responses and others are suggestive of avoidance to the ink, reminiscent of that
described by Willan (1979) for the seastar Coscinasterias calamaria contacting the ink
of Aplysia dactylomela. On sensing the ink the seastar responded by immediate cessation
of activity. In our experiment, expansion of defensive bristles by Hermodice, vigorous
grooming by Mithrax, copious production of mucus byTridachia, and temporary
cessation of heart- and scaphognathite-beating in Portunus are consistent with an
hypothesis that the ink is a sensory irritant. On this basis, Pennings’ (1994) observations
on depressed feeding of the crab Hemigrapsis sanguineus in the presence of ink of
Aplysia kurodai can be re-examined. Perhaps the lessened feeding of Hemigrapsus was
due to the ink irritating its antennary chemosensory organs (aesthetascs). This would
likely have been accompanied by increased grooming of the affected appendages as
noted with Mithrax in the present study and possibly reduction in feeding activity. In a
related study, DiMatteo (1982) showed that several types of crabs living sympatrically
with A. dactylomela, including the species Mithrax sculptus and Portunus spinimanus,
were repelled by otherwise edible pieces of fish coated with ink. From the manner in
which the crabs ‘slapped at’ and retreated from the ink, DiMatteo concluded that the ink
functioned in the sea-hare’s defense through its distasteful or noxious properties. These
observations are not incompatible with our notion of sensory irritation. In nature, a
surrounding cloud of sensory irritant might sufficiently distract a potential predator to
allow a sea hare to escape. The hypothesis that ink reduces predation by functioning as a
sensory irritant does not preclude other functions such as unpalatability, and in some
instances the same sensory devices might be involved (DiMatteo, 1981, 1982; Pennings,
1994; Nolen et al., 1995).
An hypothesis of sensory irritant is further supported by our observations on the two
fish species. During the respirometry runs, all fish responded with bouts of vigorous
swimming on initial contact with ink, behaviour which would in the field rapidly remove
T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 – 197
195
them from an ink-tainted area. Although the swimming was too brief to be reflected in
significantly higher after-ink VO 2 values for either species, it is notable that three fish
died following exposure to the highest dosages and, of these, the two puffers were fully
inflated suggestive of extreme stress. Other applications of Aplysia ink to aquatic
vertebrates have resulted in similar lethality (in blennies, Willan, 1979) or anaesthesia
(in frogs, Flury, 1915). These results point to additional properties of the ink that might
only affect vertebrates and not invertebrates, likely because of different sensory and
central nervous system structure and function in the two groups. We note, however, that
these lethal effects are likely a byproduct of other functions of the ink and caused by
forced prolonged exposure. In nature, affected organisms could likely remove themselves from the presence of ink long before any lethal effects were realized.
Our experiments were carried out in closed containers, whereas ink released in the
field would be progressively diluted following release. Our observations of ink released
in the field suggested that under conditions of moderate water motion a cloud of ink
remained dense for perhaps a minute and visible for several more minutes. We believe
that our experiments were relevant to these field conditions. Although our behavioural
experiments were carried out over a period of 5 min, qualitative observations indicated
that animals changed their behaviour within seconds of exposure to ink. Similarly, brief
bursts of swimming by fish and cessation of heart and scaphognathite beating in
Portunus all occurred less than a minute following ink addition. Thus, we suggest that in
the field brief exposure to Aplysia ink would be sufficient to distract and / or drive away
a potential predator.
Our interest in the scaling relationship of ink volume to body size in Aplyisa
dactylomela stemmed from the notion that if the ink were defensive in function, then
small individuals might be expected to produce disproportionately larger amounts of it.
This is reminiscent of certain snakes that produce more toxic venom in their young
stages (Minton, 1967; Reid and Theakston, 1978; Mackessy, 1988), and of spiders in
which more potent venom is produced in small versus large species (Quistad et al.,
1992), presumably to compensate for the smaller volumes produced. However, our
results showed an isometric relationship of ink volume to body size, with slope, b, equal
to 1. Whether the ink is more potent in the juvenile stages to compensate for the smaller
volumes produced would be an interesting subject for future study. In this regard, Willan
(1979) noted that the ink of Aplysia parvula, a species one to two orders of magnitude
smaller than A. dactylomela and living in the same habitat, was much more potent in
deterring attack by the seastar Coscinasterias calamaria than was the ink of the larger
species.
Acknowledgements
We thank Michael Haley, Director of the Discovery Bay Marine Laboratory,
University of the West Indies, Jamaica, and his staff for providing research space and
assistance during the study. Jahsen Levy and John Samuels kindly provided the portunid
crabs for the study. Funding was provided by the Natural Sciences and Engineering
Research Council of Canada in the form of a research grant to T. Carefoot.
196
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