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 (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 ®ve invertebrate species (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 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 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 Aplysia dactylomela 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 animals 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.