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Effects of sea hare ink secretion and its escapin-generated components on a variety of predatory fishes.


Predator-prey interactions can exert strong selection pressure that affects the evolution of anti-predation defenses (McClintock and Baker, 2001; Paul et al., 2007; Zimmer and Ferrer, 2007; Hay, 2009). These defenses include behavioral adaptations, body coloration, mechanical defenses, and chemical defenses. To be effective, an anti-predator defense must disrupt the predation event at the point of detection, approach, capture, or acceptance of the prey (Endler, 1986). Chemical defenses can be either passive, such as compounds constitutively found in tissues, or actively released, as in the nematocysts of a sea anemone. The adaptations are restricted by the natural history of the species, and they control the relationship that prey species have with their potential predators. Prey species that may encounter a variety of predators must be adapted for protection against a variety of predation methods and must have defenses that affect organisms with very different sensory systems and adaptations of their own.

Molluscs in general, and opisthobranch molluscs in particular, have an impressive array of defenses against a broad range of predators from diverse taxa, including sea anemones, sea stars, crustaceans, fishes, and humans (Kinnel et al., 1979; Denny, 1989; Avila et al., 1991; Cimino and Ghiselin, 2001; Cimino and Gavagnin, 2006). Opisthobranchs, which include sea hares, are soft-bodied and slow-moving benthic snails that live in many marine habitats (Carefoot, 1987; Wagele and Klussmann-Kolb, 2005). No predator is known to make a regular meal of them, but a number of generalist predators, notably fish, crustaceans, and sea anemones, have been reported in field studies to occasionally consume them (Winkler and Tilton, 1962; Pennings, 1990; Paul and Pennings, 1991; Johnson and Willows, 1999; Ginsburg and Paul, 2001; Pennings et al., 2001). Sea hares would be highly vulnerable to predators if not for the possession of a variety of defenses that include escape behaviors, large size, crypsis, and chemicals (Carefoot, 1987; Johnson and Willows, 1999). Chemical defenses of sea hares include both passive and active forms. Passive chemical defenses include deterrent and toxic molecules in the skin and other tissues that are highly effective against many predators (Winkler, 1969; Watson, 1973; Stallard and Faulkner, 1974a, b; Ambrose et al., 1979; Kinnel et al., 1979; Paul and Pennings, 1991; Pennings and Paul, 1993; de Nys et al., 1996; Pennings et al., 1999; Ginsburg and Paul, 2001; Wagele and Klussmann-Kolb, 2005; Kamiya et al., 2006; Wagele and Klussmann-Kolb, 2005; Kamiya et al., 2006; Derby, 2007), but can also include having flesh of low nutritional value (Pennings, 1990; Penney, 2002). Inking is an active chemical defense that is used as a late line of deterrence during attacks. Sea hare ink secretion is a sticky, purple mixture of the products of two glands (Nolen et al., 1995; Johnson and Willows, 1999): ink, a product of the ink gland, is a deep purple color; opaline, a product of the opaline gland, is white and highly viscous. Ink and opaline are co-secreted, mixed in the mantle cavity, and released toward the source of the attack.

Ink secretion has been shown to protect sea hares against a number of predators, especially invertebrates such as crustaceans and sea anemones, though the identity of bio-active molecules and mechanisms of its effects are largely unexplored (see reviews by Carefoot, 1987; Johnson and Willows, 1999; Derby, 2007). Mechanisms of action of sea hare ink secretion are best studied for Aplysia californica and two of its invertebrate predators, the spiny lobster Panulirus interruptus, and the sea anemone Anthopleura sola. Ink secretion reduces predation by P. interruptus through a variety of mechanisms including unpalatability, sensory disruption, and phagomimicry (Kicklighter et al., 2005; Shabani et al., 2007; Aggio and Derby, 2008). Against sea anemones, ink secretion is an unpalatable deterrent that causes tentacular withdrawal (Nolen et al., 1995; Kicklighter and Derby, 2006). Recent work on the blue crab Callinectes sapidus, has determined that one of ink's purple pigments, aplysioviolin, is a chemical deterrent (Kamio et al., 2010).

Much less is known about the effects of sea hare ink secretion on another dominant class of predators in marine habitats--predatory fishes. Ink secretion from Dolabella auricularia is unpalatable to reef fishes (Pennings et al., 1999), and ink secretion from Aplysia dactylomela induced increased swimming activity in a puffer and goby (Carefoot et al., 1999). Ink, but not opaline, from A. californica is unpalatable to the sea catfish Ariopsis felis (Sheybani et al., 2009). In fact, opaline and its amino acid fraction are appetitive to sea catfish, suggesting that opaline might contribute to the effect of the ink secretion through sensory disruption or phagomimicry (Sheybani et al., 2009).

The current study had two goals. The first was to evaluate the efficacy of sea hare ink secretion as a chemical deterrent against fish, with a future aim of examining mechanisms of its effect on this group of predators. We examined five species of fishes, which represent a variety of predation styles and habitats, since these variations might influence the effectiveness of a particular defensive strategy. The second goal was to test the deterrent effects of a set of components in ink--those produced by the escapin pathway--on these fish predators. Escapin is an L-amino acid oxidase that oxidizes its substrates, L-lysine and L-arginine in opaline, when ink and opaline are secreted simultaneously, and produces a complex set of compounds that are mild deterrents against Panulirus interruptus and Callinectes sapidus (Yang et al., 2005; Johnson et al., 2006; Kamio et al., 2007, 2009; Aggio and Derby, 2008) (Fig. 1).


Materials and Methods


To test for the effects of the Aplysia californica ink secretion on predatory fishes, we performed an ingestion assay on five species of fishes with different feeding styles, ranging from those that engulf prey whole to those that peck small pieces from larger prey items. We included in our study species that are strongly suspected of being predators of sea hares as well as some that are practical laboratory models that can be used in future mechanistic studies.

The bluehead wrasse Thalassoma bifasciatum represents a good laboratory model as well as a potential predator of the sympatric sea hare Aplysia dactylomela, and we have performed further experiments with this species to examine mechanisms of deterrent effects (Nusnbaum and Derby, 2010). Bluehead wrasses are found in the waters around Florida and the Caribbean islands (Feddern, 1965). The advantages of using this species for aquarium bioassays have been detailed previously (Pawlik et al., 1987). It is a common fish species for testing anti-predatory chemical defenses because it is easy to maintain and train to feed on artificial diets (Lindquist and Hay, 1996; Hay et al., 1998; Kubanek et al., 2000; Odate and Pawlik, 2006). For our study, juvenile animals, 5-10 cm long, in the yellow phase were wild-caught in south Florida and maintained at Georgia State University in individual 40-liter glass aquaria (50 cm X 25 cm X 30 cm) containing filtered and aerated (Whisper Filters Tetra, Blacksburg, VA) seawater (Instant Ocean, Aquarium Systems, Mentor, OH) at a salinity of 28 ppt and a temperature of about 21 [degrees]C. Fish were fed shrimp and brine shrimp ad libitum twice daily. Fish were kept on a 14:10 light/dark cycle and maintained in the same aquaria in which they were tested.

The other fishes that we tested are senorita wrasses (Oxyjulis californica), bonnethead sharks (Sphyrna tiburo), mummichogs, or killifish (Fundulus heteroclitus), and pinfish (Lagodon rhomboides). Senorita wrasses are sympatric with A. californica at intermediate depths in the Pacific (Bray and Ebeling, 1975). Although there are no records of predation events between these species, it is possible for adult senorita wrasses, which can reach 25 cm in total length, to eat juvenile sea hares. Bonnethead sharks are found along the east and west coasts of North and South America and could potentially encounter one of a number of Aplysia species including A. californica (Enric et al., 1996). This shark is a bottom-feeding predator that eats a wide variety of molluscs and crustaceans. Mummichogs are small generalist predators that typically feed on insect larvae, small crustaceans, and molluscs, and live in intertidal waterways or salt marshes throughout the Atlantic coastal areas (Bigelow and Schroeder, 1953). It is unlikely that this species would encounter a sea hare or attack one in nature, but it represents a generalist predator that can easily be trained to feed on artificial diets. Pinfish are unlikely to attack a sea hare, but they represent a predatory fish species with a variable diet and have been used in studies of the efficacy of chemical defenses (Huang et al., 2008).

Senorita wrasses, each about 15 cm long, were wildcaught by Marinus Inc. (Garden Grove, CA), shipped to our laboratory, and kept individually in aquaria in the same conditions as bluehead wrasses. Pinfish averaging 12 cm in length were obtained by dropping lines and hooks off a dock into waters near the Whitney Laboratory (St. Augustine, FL). Mummichogs were also obtained from the Whitney Laboratory; 10 cm long fish were caught in traps in shallow marshy areas. Pinfish and mummichogs were kept individually in 20-liter (40 cm X 20 cm X 20 cm) plastic containers supplied with flowing seawater and fed pieces of shrimp throughout the experiment. Bonnethead sharks were caught by personnel at Mote Marine Laboratory (Sarasota, FL) and held in that facility. The bonnethead sharks, about 20-90 cm long, were housed in a single group of 20 animals in a 227,000-liter aquarium (15 m in diameter and 3 m in depth) and fed ad libitum on a combination of shrimp and fish during an acclimation period. The acclimation period lasted until the fish fed reliably on introduced food for between 3 and 5 days for all species tested. For the assays, they were fed sparingly on shrimp to maintain hunger levels. After completion of these studies, which took 1-2 weeks, pinfish and mummichogs were returned to the waters where they were caught, and bonnethead sharks were used for further behavioral analyses by other researchers at the Mote Marine Laboratory. Senorita wrasses and bluehead wrasses were maintained for 1-3 months and tested in multiple behavioral assays before being euthanized because they could not be returned to the waters where they were caught.

Collection of sea hare secretions

Ink and opaline were collected from adult sea hares wild-caught by Marinus Inc. (Garden Grove, CA) immediately after their arrival in our laboratory. The diet of these wild-caught individuals is not known, but the presence of purple ink indicated that their diet included red algae. Secretions were collected from the dissected ink and opaline glands. Ink glands were gently squeezed to release ink. Opaline glands were centrifuged at 30,000 X g for 1 h at 4 [degrees]C to separate opaline secretion from gland tissue. Secretions collected from individual animals were pooled to reduce any effect of individual variability in contents of glands. Secretions were frozen at--80 [degrees]C until needed.

Preparation of other stimuli

Escapin, an L-amino acid oxidase in ink of A. californica, was purified from ink by using an AKTA 100 Automate fast protein liquid chromatography (FPLC; Amersham Pharmacia Biotech, Piscataway, NJ). A preparative grade Hi-load Superdex 200 16/60 column (Amersham Pharmacia Biotech) was used for initial size separation, with fractions collected in an automated fraction collector. The mobile phase consisted of 50 mmol [1.sup.-1] potassium phosphate buffer at pH 7.6. Fractions containing escapin had a yellow color and eluted separately from the purple pigments in the ink (Yang et al., 2005). To make escapin end products for L-lysine or L-lysine or 350 [micro]mol[1.sup.-1] L-arginine at 30 [degrees]C in 50 mmol [1.sup.-1] potassium phosphate buffer for 48-72 h. These are the natural concentrations of L-lysine and L-arginine found in opaline of wild-caught animals (Kick-lighter et al., 2005; Derby et al., 2007), and therefore products were tested at these maximal concentrations. Production of escapin intermediate products for lysine or arginine (Fig. 1) followed the same protocol as for escapin end product except that 4 mg/ml of catalase (C1345, Sigma-Aldrich, St. Louis, MO) was added to the solution to scavenge [H.sub.2][O.sub.2] and prevent the completion of the reaction. Escapin and catalase were removed from the solution by filtration, and the solution was lyophilized for storage at -20 [degrees]C. [H.sub.2][O.sub.2] and ammonia were tested at 145 mmol [1.sup.-1], since L-lysine is present at this concentration in A. californica ink (Derby et al., 2007) and therefore 145 mmol [1.sup.-1] is the highest concentration that [H.sub.2][O.sub.2] and ammonia could reach in a reaction. The combination of lysine intermediate products + [H.sub.2][O.sub.2] is much more bactericidal than either alone (Yang et al., 2005; Ko et al., 2008). We tested this mixture, as well as mixtures of other escapin products + [H.sub.2][O.sub.2] or ammonia, to determine if they are more effective deterrents than their components.

Ingestion assay

Preparation of pellets. Pellets were created to test the effect of added stimuli on feeding behavior, especially the acceptance of the food, as described in Hay et al. (1998) and as used previously in Sheybani et al. (2009). To make the pellets, shrimp purchased at a local seafood market were freeze-dried, then ground into a powder using a mortar and pestle. Powdered shrimp and alginate (Sigma-Aldrich) were combined in a 5:3 ratio by weight, and 8 g of this mixture was added to 100 ml of deionized water. Opaline, seawater, and uncolored escapin products were colored with 0.1% red food color (McCormick & Co., USA: listed contents are water, propylene glycol, FD&C reds 40 and 3, and propylparaben). The addition of food color to these stimuli was intended to control for the color and intensity of ink. This shrimp-alginate solution was drawn into a 50-[micro]l pipette and exuded into a 0.25 mol [1.sup.-1] CaC [l.sub.2] solution, creating a solid cylinder of 1-mm diameter that was cut into pellets 3 mm long. Unflavored alginate pellets were produced by following the same procedure except that shrimp was not added. Preliminary behavioral tests showed that shrimp-alginate pellets were attractive to fish, whereas unflavored alginate pellets were not. Shrimp-alginate pellets could be treated with test solutions by combining 1 ml of test solutions per 3 ml of alginate gel, to create test pellets. This creates pellets containing 25% full-strength test stimulus, which is likely in the range of secretion concentrations that fish are likely to encounter when attacking live, juvenile sea hares.

Behavioral testing. For bluehead wrasses, senorita wrasses, pinfish, and mummichogs, individually held animals were acclimated to hand feeding with a food stimulus, and only those fish that ate were used in subsequent testing. During the experiment, each individual of these four species was presented once with each of the 16 test substances and the control. The time between consecutive stimulus presentations was at least 20 min. Fish were tested no more than eight times each day to maintain high hunger levels. Food was fed to fish between each test, and data for a test were not used if the fish rejected or ignored the food. Alginate pellets flavored with freeze-dried shrimp powder were used in all ingestion assays except for those requiring immediate feeding after mixing of the stimuli, since the formation of the pellets requires time to gel. In these cases, 2-mm cubes of freeze-dried shrimp were treated with test substances. Using freeze-dried shrimp was especially important in tests mixing escapin intermediate products with [H.sub.2][O.sub.2], because these two products combine in a non-enzymatic reaction. The kinetics of that reaction (Kamio et al., 2009) requires that these stimuli be fed to the fish immediately upon mixing. Some unstable and transient products from the reactions are hypothesized to be involved in the deterrent effects, and they could be at undetectable levels within 1 min of mixing. Therefore, all of the experiments in which escapin intermediate products were mixed with [H.sub.2][O.sub.2] or N [H.sub.3] used freeze-dried shrimp, as did experiments with ink and opaline mixed together (ink + opaline). In these cases, the substances were applied dropwise onto the pieces of freeze-dried shrimp and immediately presented to fish. Ink and opaline were applied together in this manner, with two pipettes simultaneously releasing secretions onto the same piece of shrimp to allow mixing.

Bonnethead sharks were fed freeze-dried shrimp soaked in test substances, rather than pellets, because pellets could not be formed that would be large enough to be bite-size. Each shrimp (ca. 32 mm long) was peeled and saturated with test substances applied dropwise onto the flesh before being immediately presented to the sharks with a pair of forceps. Each of the nine test substances and the control were presented eight times to the group of 20 sharks, and each test substance was followed by a piece of food to ensure normal feeding by the sharks. At least 15 min was allowed to pass between presentations of consecutive test substances. Control and test substances were presented in a randomized order, and no more than 10 presentations were given to the sharks in a single day. The experimenter could not discriminate the identity of the sharks in the group, and thus we cannot exclude multiple treatments of a substance with the same shark.

Fish were hand-fed food held in a pair of forceps. The food items, which contained different substances as described below, were presented in a random order to avoid order effects, and they were presented blind to protect against observer bias; however, due to the deep color of ink it was not possible to completely hide its nature from the researcher. We used acceptance or rejection of food as a measure of its palatability. Acceptance is defined as taking the food into the mouth, followed by swallowing it during the test. Rejection is defined as the food not being swallowed and remaining in the aquarium at the end of the test period. When encountering a piece of food, the fish typically brought it into its mouth and flushed water through the mouth and out the gills. If the food was palatable, the fish kept the item in its mouth and swallowed it. If the food was strongly aversive, the fish either did not take it into the mouth or took it in and immediately ejected it. If the food was not strongly aversive, the fish often repeatedly brought it into its mouth and ejected it. The outcome was rated "rejection" if the food had not been swallowed by the end of the test period. The fish would generally take the food into its mouth immediately upon presentation and then would either swallow it or spit it out. The fish was observed for about 30 s after ingestion to ensure that it did not later reject a previously accepted food item. A satiety control was presented after each test sample; if the fish did not accept a control food sample, the prior response was not used in the data analysis. Responses were recorded as either "rejection" or "acceptance," and were analyzed using Cochran's Q test with post hoc testing employing one-tailed McNemar's tests.


Responses to food treated with sea hare ink or opaline

Four species of fishes--senorita wrasses, bluehead wrasses, mummichogs, and pinfish--were tested with alginate pellets of different composition. All fish were tested individually (see Fig. 2 for number of animals of each species), and all individuals used in the study accepted shrimp-flavored pellets (a positive control) and rejected unflavored pellets (a negative control). All individual fish of each species also rejected shrimp-flavored pellets containing either ink or ink + opaline, and they accepted shrimp-flavored pellets containing opaline (Fig. 2A-D). Thus, ink or ink + opaline cause significant rejection of otherwise palatable food in these four species of fish (see statistics in Fig. 2A-D).


Bonnethead sharks were tested as a single group of 20 animals rather than individually because of housing limitations. Sharks were fed freeze-dried shrimp rather than alginate pellets because pellets could not be made large enough for the sharks. The group of sharks was presented eight times with each test substance. The group accepted all eight presentations of shrimp or shrimp containing opaline (Fig. 2E). The group accepted 5 of 8 presentations (62.5%) of shrimp containing either ink or ink + opaline. Overall, there was a significant effect of treatment (Cochran's Q test, Q = 9, df = 3, P = 0.029). Pairwise testing failed to reveal a significant difference between any test substance and the control, although there was a strong but nonsignificant trend (P = 0.06) for shrimp treated with ink or ink + opaline to be rejected more than the control. Our behavioral observations revealed that those sharks that ate ink-treated shrimp handled them differently from plain shrimp: they repeatedly spat them out and took them back in their mouth before finally accepting and swallowing them.

Thus, four of the five tested species of fishes showed clear and statistically significant rejection of ink-treated food, and the other species showed a strong tendency toward rejection as well as qualitative differences in handling of ink-treated food.

Responses to food treated with escapin products

Two of the five fish species--bluehead wrasses and senorita wrasses--significantly rejected shrimp-flavored pellets or shrimp containing some of the products of escapin's activity on lysine and arginine (Fig. 3A, B). Pellets with lysine intermediate products + [H.sub.2] [O.sub.2] were rejected by 26% of bluehead wrasses and 26% of the senorita wrasses. Pellets with arginine intermediate products alone were rejected by 22% of bluehead wrasses. [H.sub.2] [O.sub.2] alone did not significantly deter feeding by any of the species tested (Fig. 3 A-E). Mummichogs, pinfish, and bonnethead sharks were not significantly deterred by any escapin products (Fig. 3C-E). Since the concentrations of escapin's intermediate and end products tested were near the theoretically highest concentrations that might occur in the secretions, these results indicate that for the two species of wrasses, escapin products are at most minor contributors to the deterrence of sea hare secretions, and for pinfish, mummichogs, and bonnethead sharks, escapin products do not contribute to the deterrence.



Animals have a diversity of defenses against predators (Endler, 1986; McClintock and Baker, 2001; Paul et al., 2007; Zimmer and Ferrer, 2007; Hay, 2009). These defenses function to disrupt the sequence of a predatory attack at the point of detection, approach, capture, or acceptance of the prey. Prey animals can utilize multiple mechanisms of protection from different predators and in different contexts (Endler, 1986). Molluscs have an impressive array of defenses to protect themselves from a broad array of predators from diverse taxa, including sea anemones, sea stars, crustaceans, fishes, and humans. Some molluscs are protected by shells, but many are not. Some, such as the squid, take advantage of speed and acute vision for protection. Chemical defenses are used extensively by both shelled and shell-less molluscs. The mucus secreted by molluscs can function as a mechanical and a chemical defense as well as a carrier for defenses (Branch, 1981; Rice, 1985; Avila et al., 1991; Ehara et al., 2002; Kicklighter et al., 2005). The skin of marine gastropods has deterrent chemicals, many of which are diet-derived (Stallard and Faulkner, 1974a, b; Pennings, 1990; Pennings and Paul, 1993; de Nys et al., 1996; Ginsburg and Paul, 2001). Mucus and deterrent-rich skin and egg masses are examples of passive defenses, but molluscs also possess a variety of active chemical defenses that are released only upon predatory attack. These chemical defenses include the ink of gastropods such as the sea hare Aplysia californica, but also include the ink of cephalopods, which may act as a visual mimic, distracter, or smoke screen in addition to its potential chemosensory effects (Caldwell, 2005; Derby et al., 2007; Wood et al., 2008).

Sea hare ink secretion as a chemical defense against a diversity of predators

Chemical defenses play a large role in the life of sea hares. Inking is a defense used only when sea hares are severely disturbed (Leonard and Lukowiak, 1985). Our observations show that sea hares will tolerate physical manipulation--for example, pecking by bluehead wrasses, poking and biting by crustaceans, ingesting by sea anemones, and handling by humans--without inking. Thus, inking is a high-threshold behavior, typically produced only in severe attacks, such as when taken into the mouth of a large fish or after vigorous pecking by smaller fishes. This high threshold would be expected if acquisition and sequestration of the active compounds in ink is energetically costly.

A. californica ink is broadly effective as a chemical defense against an array of predators. We have not found a species that does not show some aversive response to ink secretion, and many and diverse species, including cnidarians, crustaceans, and fishes, are known to be affected by external presentation of ink (DiMatteo, 1982; Nolen et al., 1995; Rogers et al., 2000). Ink is even a powerful antimicrobial agent (Ko et al., 2008) or a toxin for some animals (Flury, 1915). An animal that would otherwise be vulnerable to attack from a variety of predators must have defenses that protect it from this same variety. A chemical defense that affects the sensory systems of members of many different phyla functions as a good broad-spectrum protection.

Our study examined the use of ink by sea hares as a chemical defense against vertebrate predators, based on an ingestion assay with five species of predatory fishes: bluehead wrasses Thalassoma bifasciatum, senorita wrasses Oxyjulis californica, pinfish Lagodon rhomboides, mummichogs Fundulus heteroclitus, and bonnethead sharks Sphyrna tiburo. Our results demonstrate that sea hare ink secretion is unpalatable to all five species. All species showed aversive responses to otherwise palatable food when it was impregnated with the sea hare ink secretion (Fig. 2). This was clearest with bluehead wrasses, senorita wrasses, pinfish, and mummichogs, which significantly rejected food laced with ink. The aversion of bonnethead sharks to ink secretions was weaker but still evident, as indicated by a statistically significant effect of secretions on acceptance of food and a change in handling of food treated with ink. The lower rejection rates in the feeding assay in sharks may be explained by the fact that these experiments were performed with whole freeze-dried shrimp rather than shrimp-flavored pellets as for the other fishes. Palatability and attractiveness of potential food sources are controlled by many factors including hunger level, the presence and concentration of attractant molecules such as amino acids, the presence and concentration of deterrent molecules, and the perceived nutritional value of the food (McClintock and Baker, 2001; Cruz-Rivera and Hay, 2003). Our ability to discern finer levels of deterrence may be affected by the concentration of attractive molecules and the predation style and hunger level of the fish species.

Our test species included fish with predation styles ranging from those that would likely engulf a sea hare (bonnethead shark) to others that would likely attack a sea hare by pecking small pieces from it (wrasses, pinfish, mummichogs). Some are more likely than others to be predators of sea hares (bonnethead sharks, wrasses, pinfish) (Bigelow and Schroeder, 1953; Bray and Ebeling, 1975; Enric et al., 1996; Huang et al., 2008). Some are proven behavioral models in studies of chemical defenses and good candidates for future physiological mechanistic studies (bluehead wrasses) (Pawlik et al., 1987; Lindquist and Hay, 1996; Hay et al., 1998; Kubanek et al., 2000; Odate and Pawlik, 2006).

Similar effects of sea hare ink secretion on food acceptance were observed with sea anemones (Nolen et al., 1995; Kicklighter and Derby, 2006), spiny lobsters (Kicklighter et al., 2005; Aggio and Derby, 2008), crabs (DiMatteo, 1982), reef fishes (Pennings et al., 1999), sea catfish (Sheybani et al., 2009), and seagulls (DiMatteo, 1981). Together, these results demonstrate that ink secretion is unpalatable to a broad array of marine predators.

Chemical defenses, such as sea hare ink, can have effects on different phases of attack by predators. The process of predatory attack involves two phases: approach and capture of food, when the prey is taken into the mouth; and acceptance, when the prey is swallowed and consumed (Endler, 1986; Ritson-Williams and Paul, 2007). When ink is presented as a cloud, as might happen before a predator actually bites or attempts to ingest sea hares, it can cut off an attack (Nolen et al., 1995; Kicklighter et al., 2005; Nusnbaum and Derby, 2010). When presented in food, as might happen when a predator takes a bite of a sea hare and simultaneously gets a mouthful of ink, it causes egestion (DiMatteo, 1982; Rogers et al., 2000; Kicklighter et al., 2005; Nusnbaum and Derby, 2010). In bluehead wrasses, these varied effects are due to responses by the olfactory system and the gustatory system respectively (Nusnbaum and Derby, 2010). Understanding how a potential chemical defense is detected by the predators' sensory systems gives insight into both the co-evolution of these signals and the sensory biology of deterrence. There are many examples of plant chemical defenses against insects and the identity of the insects' sensors that detect them (e.g., Bernays et al., 1989; Glendinning et al., 1990; Stowe et al., 1995). For example, some tannins produce deterrent effects on herbivores, mediated by taste receptors on mouthparts, and at high concentrations tannins can produce systemic toxicity (Mueller-Harvey, 2006). A herbivore's detection of

deterrent compounds and association of this effect with the tannin source can help it to avoid toxic effects and protect the tannin producer from predation. Alternatively, toxic or aversive plants can produce volatile substances (which may or may not be directly linked with the toxic effects) that herbivores may associate with the somatosensory or gustatory experiences, causing them to avoid such defended prey (Woolfson and Rothschild, 1990; Rothschild et al., 1994). Sea hare ink may both protect the prey from immediate predation and, by stimulating learned aversion in the predator, convey future protection (Long and Hay, 2006).

Identity of the components in sea hare ink secretion that are deterrents against fish

Sea hare ink secretion is a mixture of ink from the ink gland and opaline from the opaline gland. When combined, ink and opaline form a more persistent, sticky secretion than ink alone. To determine whether the defensive chemicals in ink secretion are present in ink, opaline, or some of the identified components of the ink secretion, we used the same five species of predatory fishes. We show that it is ink, not opaline, that is highly unpalatable (Fig. 2). When these two secretions combine, at least one enzyme and its substrate are combined: escapin in ink is mixed with high concentrations of L-lysine and L-arginine in opaline (Yang et al., 2005; Johnson et al., 2006). There are likely other compounds formed by the mixing of the two secretions, which may contribute to the efficacy of ink. So far, escapin compounds have been tested on several species of predators, and they have proved to be relatively unimportant contributors to overall deterrence. Escapin's reaction products, which constitute a complex mixture (Fig. 1; Kamio et al., 2009), had limited effects on the palatability of food for our test fishes (Fig. 3). For senorita wrasses and bluehead wrasses, shrimp containing a mixture of lysine intermediate products and [H.sub.2][O.sub.2], which are products of escapin's activity on lysine, was rejected significantly more than plain shrimp, though rejected less than shrimp containing ink secretion. This mixture of lysine intermediate products and [H.sub.2][O.sub.2] is also responsible for the secretion's powerful bactericidal effects (Yang et al., 2005; Ko et al., 2008). Blue crabs and spiny lobsters are also deterred by the high levels of [H.sub.2][O.sub.2] released during the enzyme-catalyzed reaction (Aggio and Derby, 2008; Kamio and Derby, unpubl. data). Although escapin reaction products are probably not the major deterrents against species that have been tested, they may contribute to the overall effectiveness of the secretion and may be maximally effective against other predators.

Thus, having a defensive secretion composed of many active compounds is useful for a species that is potentially so vulnerable to so many predators. Some compounds may be fairly specific to certain predators, so the prey species may benefit from possessing many compounds of diverse functional types. Other compounds may be broadly effective, such as [H.sub.2][O.sub.2] or phagomimetic levels of amino acids (Kicklighter et al., 2005). The molecular identities of the compounds accounting for most of the unpalatability of ink to any predatory species are mostly unknown, though the purple pigment aplysioviolin has recently been identified as being effective against both invertebrate and fish predators (M. Kamio, T. V. Grimes, M. H. Hutchins, R. van Dam, and C. D. Derby, unpubl. data). This complement of chemical defenses, combined with other (nonchemical) defenses, results in a well-defended animal.


We thank Cynthia Kicklighter for preliminary experiments; Michiya Kamio and Juan Aggio for help with extracting ink and opaline; Ko-Chun Ko for help with purifying escapin; Joan Maor for experimental assistance; Henry Feddern of Tavernier, Florida, for collection of bluehead wrasses; and Robert Hueter and Jack Morris of Mote Marine Laboratory and Peter Anderson and Barry Ache of the Whitney Laboratory for providing animals and facilities. The care and use of fish was approved by the Institutional Animal Care and Use Committee at Georgia State University, under protocols A06039-0079 and 2007-0030. Funding was provided by NSF IBN-0614685, a GSU Brains & Behavior fellowship, and support from the NSF IGERT program at Georgia Institute of Technology.

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Received 17 July 2009; accepted 12 February 2010.

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Author:Nusnbaum, Matthew; Derby, Charles D.
Publication:The Biological Bulletin
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Geographic Code:1USA
Date:Jun 1, 2010
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