Journal of Experimental Marine Biology and Ecology, L 234 (1999) 185±197

A test of novel function(s) for the ink of sea hares

Thomas H. Carefoota,* , Steven C. Pennings b , Jean Paul Danko a aDepartment of Zoology, University of British Columbia, 6270 University Blvd, Vancouver, BC V6T 1Z4, Canada bMarine 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 (: ) 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 dactylomela (Rang), none of ®ve invertebrate (including A. dactylomela) or two ®sh species signi®cantly altered their oxygen uptake, and neither of two crab species signi®cantly altered their heart and/or scaphog- nathite 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 ®sh species was strongly affected by ink, supporting our hypothesis that the ink functions as an irritant. Observed behavioural changes included bristle erection by ®reworms, 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 ®sh. Similar behavioural changes by potential predators would likely lead to reduced predation rates on Aplysia spp. in the ®eld. 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 (speci®cally, 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)00153-1 186 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 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 contrac- tions and parapodial ¯appings 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 ®eld, 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 ®eld, in this case an apparently under attack from the star®sh Coscinasterias calamaria. Indeed, predators of Aplysia species are essentially unknown (for review see Carefoot, 1987; Pennings, 1990a). Apart from ink as a possible ®rst 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 conspeci®cs (Fiorito and Gherardi, 1990; Stopfer et al., 1993; this last idea is consistent with the notion that cephalopod ink may function as an intraspeci®c alarm substance: Gilly and Lucero, 1992, and others). However, based on the observation of Willan (1979) that locomotory movement of the star®sh Coscinsterias calamaria was retarded by contact with sea-hare ink, additional hypotheses of metabolic depressant- effect 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 ®sh 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 such as crabs and ®sh 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 ¯ow-through seawater system with an abundant supply of red algae as food. Additionally, several species of other invertebrates and ®sh were collected from the Aplysia habitat and kept in the laboratory seawater tanks (the predatory ®reworm 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 abun- dance, taxonomic diversity, and ease of collection and maintenance in the laboratory. However, we note that the majority of the taxa selected (®sh, 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 suf®cient ink for a number of experiments. The ink was kept at 58C between 188 T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 ±197 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 ®ngerbowls 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 speci®c 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 ®ve large resting adult Aplysia and the same volume of seawater into the siphons of ®ve 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 (V ) before and after exposure to ink was measured in the same O 2 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 insuf®cient numbers could be found. In addition, two potential ®sh 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 ¯oor 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 O2 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. 21 Oxygen uptake was measured as mg O2 ?g live animal at 288C and 32½. To eliminate size as a factor in the statistical analyses, all measured V values for a particular species O 2 were converted to equivalent rates for a `standard animal' of average adult mass of that species using

V (x mass (g)) 5 (x/exp. mass (g))b ?V (exp.), OO22 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 V against body mass for each test species). A typical run consisted of selecting an O 2 animal and placing it in an appropriate-sized respirometer. Fresh seawater was run through the respirometer until the animal became quiescent, after which baseline V was O 2 measured over a time suf®cient to give a signi®cant negative slope of oxygen concentration (P ) over time (usually 15±20 min). Following this, a known amount of O 2 freshly collected ink was injected into the respirometer with an hypodermic syringe, and V recorded for a further 15±20 min. Actual duration of runs varied with the type and O 2 size of animals, but at no time was P allowed to drop below 70% saturation level. O 2 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, 190 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 scaphog- nathite were similarly analysed with R-MANOVA using time as the main factor. Signi®cance 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 signi®cantly related to sea-hare size (log Vol522.0411 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 mgml21 oran 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 signi®cant 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 signi®cantly less, and were signi®cantly more likely to expose their bristles and adopt a contracted body posture. Mithrax's locomotion was unaffected (Fig. 1b), but they groomed signi®cantly more and exhibited signi®cantly fewer feeding movements in the presence of ink. Echinometra (Fig. 1c) showed signi®cantly 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 signi®cantly higher in the presence of ink. Qualitative observations on the opisthobranch Tridachia crispata showed that at low concentrations (,0.06 mg ml21 ), ink stimulated initial ¯uttering of the parapodia that quickly subsided, while at high concentrations (.0.15 mg ml21 ) 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 ®ve 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 signi®cant 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 192 T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 ±197 as ink-treated versus control. Ink alone in the respirometer showed less than 2% reduction in P over the normal duration of an experiment (about 30 min). A common O 2 feature of all respirometry runs was that V decreased from the `before' to `after' parts O 2 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 V was signi®cant only for the goby, Gnatholepis (Table 1; F59.92, O 2 P50.005, R-MANOVA), but neither treatment (ink vs water; F50.37, P50.55), nor the interaction of treatment and time (before vs after) for this species was signi®cant (F50.09, 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 signi®cant. We conclude from this part of the study that sea-hare ink does not signi®cantly affect oxygen consumption of these particular invertebrates and vertebrates over the time-periods and concentrations chosen. Neither the puffer nor goby showed signi®cant effects of ink on V over a O 2 concentration range of 0.02±0.20 mg ml21 . However, at the high end of the range, two puffers and one goby died a few hours after the respirometry experiment had ®nished. Both puffers were fully in¯ated prior to death, suggestive of stress. Heart rates (Mithrax and Portunus) and scaphognathite rates (Portunus only) showed small but non-signi®cant 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 signi®cant effect on heart rate in either species, nor on scaphog- nathite 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 (V ) of various invertebrates and vertebrates treated with sea-hare ink or seawater O2 (control) Standard N Ink N Seawater size V before V after % Diff. V before V after % Diff. OO22 OO 22 (live g) Hermodice 5 21 0.6660.36 0.5260.37 221 22 0.8060.52 0.5860.21 228 Mithrax 1.25 26 0.4360.17 0.3860.18 212 23 0.4960.21 0.3560.17 229 Echinometra 30 22 0.3260.18 0.2760.24 216 25 0.2960.17 0.2660.13 210 Tridachia 2.5 21 0.3760.31 0.2960.13 222 15 0.3160.16 0.1760.09 245 Aplysia 30 24 3.461.2 2.761.2 220 23 3.461.3 3.161.1 212 Diodon 10 6 6.962.7 5.562.6 220 6 7.864.1 5.962.7 224 Gnatholepis 2 7 0.9060.61 0.4760.27 248* 7 1.0760.30 0.6660.17 238* Values for V are mean mg O ?indiv21 ?h(21 6S.E.) for the idicated number of individuals of the `standard O22 size' shown (see text for details). The asterisks indicate signi®cant difference between `before' and `after' rates within a given treatment F59.92, 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. 194 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 signi®cant 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 ®nd 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 ®sh 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 suf®ciently 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 ®sh species. During the respirometry runs, all ®sh responded with bouts of vigorous swimming on initial contact with ink, behaviour which would in the ®eld 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 re¯ected in signi®cantly higher after-ink V values for either species, it is notable that three ®sh O 2 died following exposure to the highest dosages and, of these, the two puffers were fully in¯ated 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 them- selves 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 ®eld would be progressively diluted following release. Our observations of ink released in the ®eld 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 ®eld 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 ®sh and cessation of heart and scaphognathite beating in Portunus all occurred less than a minute following ink addition. Thus, we suggest that in the ®eld brief exposure to Aplysia ink would be suf®cient 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 T.H. Carefoot et al. / J. Exp. Mar. Biol. Ecol. 234 (1999) 185 ±197

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