Journal of Chemical Ecology, Vol. 23, No. 3, 1997

BEHAVIORAL AND CHEMICAL DEFENSES OF MARINEPROSOBRANCH GASTROPOD Calliostoma canaliculatum

IN RESPONSE TO SYMPATRIC SEASTARS

PATRICK J. BRYAN,' JAMES B. MCCLINTOCK,1 * andMARK HAMANN2

Department of Biology, University of Alabama at BirminghamBirmingham, Alabama 35294

2Department of Pharmacognosy, University of MississippiUniversity, Mississippi 38655

(Received March 28, 1996; accepted October 16, 1996)

Abstract—The gastropod Callioxtoma canaliculatum displays a series ofaggressive escape behaviors upon contact with tube feet of the predatoryseastars Pycnopodia helianthoides and Pisaster giganteus. Escape behaviorsare predator specific. Calliostoma canaliculatum moves away from contactwith P. giganteus more frequently than P. helianthoides, clamping down withthe foot or retracting the head and foot into the shell when exposed to P.helianthoides. If escape from the grasp of either seastar tails, C. canaliculatumreleases a yellow-colored exudate from the hypobranchial gland and subse-quently retracts both the head and foot fully into the shell. This exudatecontains noxious compound(s) as evidenced by retraction of tube feet andarms away from the exudate in both seastars. Tube-foot retraction responsesto dilutions of the exudate indicates that both species of seastars are able todetect the exudate at a concentration of 3.2 x 10' mg exudate/ml seawater.Pisaster giganteus is more responsive to the exudate than Pycnopodia helian-thoides, moving away from the source as well as retracting the tube feet andarm. Snails spread the exudate over their shells with their foot, perhaps toensure defense from predators for some time period after exudate release. Theexudate was collected and extracted in chloroform-ethyl acetate (1: 1), thenfractionated using flash chromatography. The most bioactive fraction, as evi-denced by tube-foot retraction, was soluble in ethyl acetate and appeared tocontain two major compounds.

Key Words—Gastropod, predation, chemical secretion.

*To whom correspondence should be addressed.

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0098 0331/97/0300-0645$l2.50/0 © [997 Plenum Publishing Corporation

646 B R Y A N , MCCLINTOCK, AND H A M A N N

INTRODUCTION

Many studies describe the escape responses of marine gastropods to livepredators (Gore, 1966; Dayton et al., 1977; Phillips, 1977; Hoffman et al.,1978; Harvey et al., 1987; Miller, 1986) and predator odors (Mackie et al.,1968; Mackie, 1970; Harvey et al., 1987; Duval et al., 1994). Most marinegastropods display similar suites of avoidance and escape behaviors. Some spe-cies possess the ability to detect predator odors at a distance (Dayton et al.,1977; Harvey et al., 1987). These gastropods often alter their direction of loco-motion and begin to move quickly away from the odor source (Feder, 1963,1967; Mackie, 1970; Phillips, 1977). Escape behaviors are displayed upon con-tact with a potential predator. Many gastropods display a "mushrooming"behavior and/or violently rotate their shells back and forth (Weldon and Hoff-man, 1979; McClintock, 1985). Calliostoma ligatum has been observed to turnand "bite" seastar predators before fleeing (Harrold, 1982).

Another adaptation common in sessile or sluggish marine invertebrates toreduce predation is the use of chemical defense mechanisms (reviewed by Bakuset al., 1986; Paul, 1992; Pawlik, 1993). Secondary metabolites that make tissuesunpalatable to predatory fish and invertebrates have been identified in marinesponges (Walker et al., 1985; McClintock, 1987; McClintock et al., 1994),ascidians (Paul et al., 1990; Lindquist and Fenical, 1991), soft corals (Wylieand Paul, 1989; Fenical and Pawlik, 1991; VanAlstyne and Paul, 1992; Slatteryand McClintock, 1995), and opisthobranch and prosobranch gastropods(reviewed by Faulkner, 1992). The majority of these marine invertebrates relyon sequestering defensive chemicals in their tissues to deter potential predators(Pennings, 1994). Chemical antipredator defenses among the Gastropoda havefocused on the opisthobranchs (Faulkner, 1992). As many opisthobranchs lacka protective shell, they are particularly vulnerable to predation and must rely ondefensive chemistry and crypsis for their protection.

Prosobranch gastropods possess a calcified shell, which is assumed to bean effective barrier against predation (Norton, 1988). Nonetheless, many pro-sobranch gastropods are significant prey for fish (Norton, 1988), other gastro-pods (Menge, 1974; Ansell and Morton, 1987), and echinoderms (Harrold,1982; Paine, 1969, 1974). Avoidance behaviors of prosobranch gastropods topredatory seastars have been investigated (Feder, 1963, 1967; Philips, 1976,1977). The ability of gastropods to flee from seastars or withdraw into the shellare believed to be the predominant methods of defense against predation (Feder,1963, 1967).

When foraging on the benthos, juvenile and adult Calliostoma canalicu-latum are vulnerable to predation by the large, highly mobile, seastar Pycno-podia helianthoides. While gastropods make up approximately 80% of the dietof P. helianthoides, C. canaliculatum represents only 1.5% of this total (Herr-

SNAIL ANTIPREDATOR DEFENSE 647

linger, 1980). Calliostoma canaliculatum has been observed to release a brightlycolored yellow exudate from its mantle following contact with P. helianthoides(Watanabe, personal communication), suggesting that this released exudate couldbe a defensive response to predation. The objectives of the present study wereto: (1) determine if C. canaliculatum can detect waterborne odors from predatoryseastars, (2) describe the behavioral escape response of C. canaliculatum to thepredatory seastars P. helianthoides and P. giganteus, (3) determine if C. canal-iculatum differentiates between predatory seastars, (4) record the reaction ofpredatory seastars to the exudate released by C. canaliculatum, and (5) chem-ically fractionate the exudate utilizing flash chromatography to isolate the activecompound(s).

METHODS AND MATERIALS

Collections. Gastropods and seastars were collected in August 1995 at 10m depth from Macrocystis pyrifera beds in Stillwater Cove, Monterey, Cali-fornia. Gastropods and seastars were placed in ambient seawater and shippedseparately to the University of Alabama at Birmingham. Immediately upon arrivalthey were placed in separate holding tanks equipped with recirculating filtrationand containing unfiltered artificial seawater (35 ppt) held at 10-12°C. Bladesof M. pyrifera were placed in tanks with gastropods as a food source. Theseastars Pycnopodia helianthoides and Pisaster giganteus were fed the gastropodTegula funebralls.

Gastropod Responses to Seastar Odor. Individual gastropods were placedin one of 60 sterile plastic containers (10 cm long x 10 cm wide X 30 cm tall)with 200 ml of artificial 0.45-/*m-filtered seawater (ASW). Fresh ASW wasused for preparation of all bath water and in all assays. Water used in an assaywas not used again in any part of the study. After allowing individuals to adjustto the container for a period of 1 min, 200 mi of ASW (control) or 200 ml ofseastar bath water was added to the containers such that 30 control and 30experimental treatments were conducted for each species of seastar odor. Bathwater was prepared by submerging whole seastars of each species in ASW at aratio of 200 g of seastar per liter of seawater for a period of three hours. Aftera period of 2 min, the height each gastropod had climbed up the side of thecontainer was measured to the nearest mm. Bath water of both species of seastarwas prepared and tested in the same fashion.

Contact with Seastar Tube Feet. Five Pisaster giganteus and five Pycno-podia helianthoides were maintained in separate seawater aquaria. Tube feetfrom randomly selected individuals were removed with tweezers from the aboralsurface of the arm and immediately employed in an avoidance trial. ThirtyCalliostoma canaliculatum were held in individual glass crystallizing dishes (15

648 BRYAN, MCCliNTOCK, AND HAMANN

cm diameter X 7.5 cm deep) containing 200 ml of ASW. Gastropods wereallowed to adjust to the dishes for a period of at least 15 min prior to eachavoidance trial. Each trial consisted of placing a tube foot held in a pair offorceps in contact with the mantle tissue of a gastropod. Responses were class-ified into a hierarchy where level 0 = no reaction; level 1 = movement awayfrom the tube foot; level 2 = movement away and shell twisting; level 3 =movement, twisting and exudation of exudate; level 4 = movement and exudaterelease; level 5 = withdrawal into the shell and clamping down; level 6 =withdrawal and exudate release. Contact trials were performed on 30 individualsnails, with each snail exposed to both tube feet from P. helianthoides and P.giganteus. Controls consisted of contacting gastropods with the forceps aloneand with a cotton swab alone. A Wilcoxon signed-rank test was performed tocompare levels of responses between seastars. The numbers of gastropods releas-ing exudates in response to each seastar species were compared with a Student'st test (a = 0.05).

Seastar Responses to Gastropod Exudate. Gastropods were induced torelease exudate by contacting them with seastar tube feet. The exudate wascollected in a Pasteur pipet as it was expelled from the hypobranchial gland.The seawater/exudate mixture was transferred to a 20-ml glass vial and held at2°C. The seawater/exudate mixture was then desalted by passing it through anAmberlite XAD-2 column (Quinn et al., 1980). Collected material was driedand then weighed to approximate the concentration of exudate in seawater as itis expelled (the exudate becomes increasingly dilute as it diffuses away fromthe gastropod; baseline concentrations were determined to calculate appropriateconcentrations for bioassay). The exudate was then resolubilized in ASW to theoriginal concentration and tested in a behavioral assay at concentrations of 3.2,0.32, 0.032, 0.0032, and 0.00032 mg/ml seawater. Behavioral assays consistedof placing individual seastars, either Pycnopodia helianthoides or Pisastergiganteus (N = 5 of each) in plastic bowls (30.5 cm diameter X 15.2 cm deep)containing 2 liters/ASW. Each seastar was tested five times, with a differentarm tip being utilized in each trial. Seventy-five microliters of seawater/exudatemixture was released approximately 1 cm from the tip of the arm. The level ofresponse was separated into one of four hierarchical categories: level 0 = noresponse; level 1 = tube-foot withdrawal (TFW); level 2 = TFW and closingof the ambulacral groove; level 3 = TFW, tightening, and coiling of the arm;and level 4 = TFW, tightening, coiling, and movement away from the exudate.In addition, overall time of sustained tube-foot withdrawal and arm coiling inresponse to the exudate or a seawater control was recorded to the nearest second.Levels of responses to exudate and seawater controls were compared using aWilcoxon signed rank test. Times of sustained tube-foot withdrawal and armcoiling in response to exudates or seawater controls were analyzed with a Stu-dent's t test (a = 0.05).

SNAIL ANTIPRBDATOR DEFENSE 649

Fractionation of Exudate. Exudate was collected from gastropods by induc-ing the release through contact with a tube foot of either Pisaster giganteus orPycnopodia helianthoides. Forty milliliters of seawater/exudate was partitionedtwice in a separatory funnel with a 3x volume of chloroform; then the mixturewas partitioned twice with ethyl acetate (3x volume). The ethyl acetate andchloroform extracts were combined to yield a lipophilic exudate extract anddried under rotary evaporation at 35°C. The dried extract (95.6 mg) was re-solubilized in ethyl acetate and loaded onto a flash column of silica 60 A,300-400 mesh. Sequential solvent systems ranging from very nonpolar to polar(100% hexane; 8:2 Hex-ethyl acetate; 4:6 Hex-EtOAc; 2:8 Hex-EtOAc; 9: 1EtOAc-methanol; 6:4 EtOAc-MeOH; 3 :7 EtOAc-MeOH; 100% MeOH) wereemployed and each fraction collected. The collected fractions were spotted ontosilica TLC plates (Whatman Silica Gel 60 A; 250 pm thickness) and run withvarious solvent systems to optimize separation. Plates were visualized by spray-ing the plate surface with 50% sulfuric acid and heating. Fractions that containedcompounds/mixtures with similar Rt values were combined and the resultant fivefractions were reassayed using the same techniques as described for the originalexudate. Four concentrations (3.2, 0.32, 0,032, and 0.0032 mg/ml seawater)were tested for each fraction.

RESULTS

The climbing response of gastropods exposed to the waterbornc odors ofPisaster giganteus and Pycnopodia helianthoides did not differ significantly fromthe seawater controls (P = 0.356, Wilcoxon). Forty-nine percent of the gastro-pods (N = 30) exposed to the control filtered seawater climbed the sides ofcontainers a mean distance of 9.2 cm. Fifty-four and 40% of the gastropodsclimbed the containers when exposed to the waterborne odors of P. helianthoidesand P. giganteus, respectively. The mean distance climbed by gastropodsexposed to waterborne odors of P. helianthoides and P. giganteus were 8.5 and9.1 cm, respectively.

Behavioral responses of C. canaliculatum to contact with the tube feet ofthe two species of seastars were significantly different (P < 0.001, Wilcoxon).Gastropod responses to contact with the tube feet of P. giganteus were primarilylevel 3 and 4 responses. A very low percentage of snails (7%) displayed a level6 response to P. giganteus, whereas 50% displayed a level 6 response to P.helianthoides (Figure 1). Additionally, 90% of snails contacted by P. helian-thoides tube feet released exudate, whereas 75% released exudate in responseto P. giganteus tube feet.

Exudate tested at a concentration of 3.2 mg/ml seawater caused a mean

650 BRYAN, MCCLINTOCK, AND HAMANN

FIG. 1. Calliostoma canaliculatum response to seastar tube-foot contact. Levels ofresponse are indicated by numbered ranks: 0 = no reaction, 1 = movement away fromtubefoot; 2 = movement away and shell twisting; 3 = movement away, shell twisting,and exudation; 4 = movement away and exudation; 5 = withdrawal into shell; 6 =withdrawal and exudation. No response was observed for contact with forceps or cottonswabs (mechanical controls). Bars represent responses of 40 individual snails assayed.

tube-foot retraction time of 51.7 and 101.2 sec for P. helianthoides and P.giganteus, respectively (Figure 2). Arm withdrawal times were longer than tube-foot retraction, averaging 76.2 and 122.1 sec for P. helianthoides and P. gigan-teus, respectively, at 3.2 mg exudate/ml seawater. Both species of seastar wereinsensitive to exudate at a concentration of 3.2 X 10 4 mg/ml seawater. The

SNAIL ANTIPREDATOR DEFENSE 651

FIG. 2. Tube-fool and arm withdrawal responses of Pycnopodia helianthoides and Pixas-ter giganteus to exudate of Calliostoma canaliculatum. The arm withdrawal response ofboth species of seastar lasted longer and was initiated at lower concentrations than thetube-foot withdrawal response. Asterisks indicate a significant (P < 0.05, Student's ttest) difference in the mean retraction time in comparison to the control, for each con-centration. Data plotted are mean ± standard deviation of 30 replicates.

652 BRYAN, MCCLINTOCK, AND HAMANN

FIG. 3. The level of response to Calliostoma canaliculatum exudate for Pycnopodiahelianthoides and Pisaster giganteus. The level of response (y axis) were scored bynumerical ranks: 0 = no response; 1 = tube foot (TF) withdrawal and tightening of armtip around ambulacral groove; 2 = TF withdrawal and tightening of arm tip aroundambulacral groove; 3 = TF withdrawal, tightening, and arm coiling; 4 = TF withdrawal,tightening, arm coil, and movement away from source of mucous. Data plotted are mean± standard deviation of 30 replicates.

mean level of response of P. giganteus (3.8 of 4) at 3.2 mg/ml seawater washigher than that of P. helianthoides (2.8 of 4) (Figure 3). The level 4 responseis the most extreme, indicating tube-foot retraction, arm withdrawal, tighteningof the ambulacral groove, and directional movement away from the exudate.The mean response of both species of seastar to the control (seawater) was alevel of 0.2 response, indicating slight tube-foot retraction.

The fractionation of the exudate and reassaying of the fractions indicatedthat the active component(s) were present in fraction 3 (Figure 4). These com-

SNAIL ANT1PREDATOR DEFENSE 653

FIG. 4. A: Thin layer chromatography visualization of flash chromatography separatedfractions of Calliostoma canaliculatum exudate. The solvent system was 3:2 ethylace-tate-hexane. B: Response of the seastar Pisaster giganteus to fractions of C. canalicu-latum exudate. Asterisks indicate a significant (P < 0.05, Student's t test) difference inthe mean retraction time in comparison to the control. Data plotted are mean ± standarddeviation of 10 replicates.

654 BRYAN, McCLINTOCK, AND HAMANN

pounds were eluted with a 40% hexane-60% ethyl acetate solvent system. Thehigher eluting of the two spots in fraction 3 appeared yellow in color. Furtherpurification and reassaying of compounds from the active fraction have revealedthat two compounds (A and B) cause mean tube-foot retraction times of 31 and28 sec, respectively, in P. giganteus.

DISCUSSION

The lack of escape behaviors in Calliostoma canaliculatum in response toodors from predatory seastars indicates escape is initiated only upon contactwith the tube feet. Escape behavior typically is initiated by movement awayfrom the area contacted by the tube feet, followed almost immediately by shelltwisting. Occasionally gastropods will also retract into the shell. The release ofthe yellow exudate appears to be a final tactic to escape from the grasp of apredatory seastar. C. canaliculatum may release exudate while fleeing or whileretracting their head and foot into their shells. The initial escape behaviorsexhibited by C. canaliculatum are classic gastropod escape responses describedin many previous studies (Feder, 1963; Phillips, 1977). However, the releaseof a defensive substance from the hypobranchial gland is rare among mollusks.The release of ink as a camouflaging defense and potential chemical irritant isknown in sea hares (DiMatteo, 1981, 1982; Nolen et al., 1995) and cephalopods(Barnes, 1987). Ink release differs somewhat from the behavior observed in C.canaliculatum, since the exudate from C. canaliculatum is strictly chemodefen-sive in nature. Aplysia californica ink acts primarily to visually confuse potentialpredators (DiMatteo, 1982; Nolen et al., 1995) but may also be distasteful(DiMatteo, 1981). In squid, release of ink forms a dummy squid to divert apredator's attention. However, alkaloids in the ink may also be repellent topredators and/or anesthetize their chemoreceptors (Barnes, 1987). Several gas-tropods including Cirostrema sp., Nucella lapillus, Ocenebra erinacea, Clathrussp., and Ianthina janthia release a purple secretion from their hypobranchialgland (Fretter and Graham, 1962). This purple dye contains dibromoindigo,which is produced once colorless chromogens in the exudate are exposed tooxygen and light (Fretter and Graham, 1962). The release of dye in these gas-tropods could serve a defensive function; however, the ecological significanceof these compounds has yet to be investigated.

Calliostoma canaliculatum attempted to flee following contact with Pisastergiganteus more frequently than in response to contact with Pycnopodia helian-thoides. In contrast, gastropods retracted into their shells more often when con-tacted with tube feet of P. helianthoides than P. giganteus, and higherpercentages of gastropods released exudate when provoked by P. helianthoides.These striking differences in the responses of C. canaliculatum to contact with

SNAIL ANTIPREDATOR DEFENSE 655

tube feet of these two seastars indicates that individuals may be able to employcontact chemoreception to detect differences in seastar metabolites (Phillips,1978). Investigations of the secondary metabolite chemistry of P. helianthoidesand P. giganteus indicate both species produce a unique variety of water-solublesaponins and polyhydroxy steroids (Bruno et al., 1989; Zollo et al., 1990).Several studies have determined that gastropods can differentiate between odorsof different predatory seastars (Phillips, 1976), crab predators (Alexander andCovich, 1991; Duval et al., 1994), and detect the dietary condition of a predator(Feder, 1963; Duval et al., 1994). A similar type of chemical recognition couldbe involved in C. canaliculatum. One possible explanation for the differentresponses shown by C. canaliculatum to contact with both seastars is related tothe relative speeds at which P. helianthoides and P. giganteus move. Pycno-podia helianthoides moves at a much faster rate than P. giganteus (P. Bryan,personal observation). It may be possible for C. canaliculatum to flee effectivelyfrom the sluggish P. giganteus, whereas individuals would be easily capturedby the more rapid P. helianthoides.

Both species of seastars showed avoidance behaviors to the yellow exudatereleased by C. canaliculatum. The exudate was active at retracting seastar tubefeet at a concentration as low as 3.2 x 10^2 mg/ml seawater. It is likely thatthe exudate itself or active compounds in the exudate entail some energetic costfor the snail to produce. It would not be advantageous to release energeticallyvaluable defensive material without just cause. The cost of chemical defensehas been extensively studied in terrestrial plants (Rhoades and Cates, 1976;Coley et al., 1985). Recent studies suggest that similar investments and trade-offs are made in marine invertebrates as in plants (Hay and Fenical, 1988).

Based on analysis by thin-layer chromatography, there are at least twomajor compounds present in the bioactive fraction of the exudate. These non-polar compounds are soluble in ethyl acetate and relatively insoluble in seawater.The use of water-insoluble exudate to contain these compounds seems to be aneffective adaptation to ensure retention of compound efficacy as long as possible.Moreover, C. canaliculatum was observed to spread the exudate over its shellwith its foot. This behavior would place the defensive compounds on the surfaceof the shell where they would be detected upon contact by a predatory seastar.Water-insoluble compounds are also advantageous in that they would diffuseslowly into the seawater, ensuring that the potency of the exudate will remainintact for some period. Future analyses will attempt to reveal the chemicalstructure of these compounds. Estimates of energetic cost can then be determinedonce metabolic pathways have been identified.

Acknowledgments—We would like to thank Dr. D. Rittschof and two anonymous reviewersfor their valuable comments on this manuscript. We would also like to thank J. Heine and D. Carneyof Moss Landing Marine Laboratories for assistance in collection of gastropods and seastars. This

656 BRYAN, MCCLINTOCK, AND HAMANN

research was supported by the Letner Gray Fund for Marine Research grant to P.J.B. and NSFEPSCoR grant EHR 9108761 to J.B.M.

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