JOURNAL OF EXPERIMENTAL MARINE BIOLOGY

Journal of Bxnerimental Marine Bioloev and Ecoloev. AND ECOLOGY -, ELSEVIER 210 (1997) 173-186--

Structural and chemical defenses of echinoderms from the northern Gulf of Mexico

Patrick J. Bryana’*, James B. McClintockb, Thomas S. Hopkins”

“Department of Biology, Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong

“Department of Biology, Universiry of Alabama at Birmingham, Birmingham, AL 35294, USA ‘Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 3.5487, USA

Received 6 June 1995; revised 6 May 1996; accepted 10 June 1996

Abstract

The feeding deterrent effects of echinoderm body-wall tissues and ethanolic extracts containing mid-polarity compounds were evaluated utilizing generalist fish and crabs as model predators. The body-wall tissues of the echinoderms examined ranged IO-fold from 0.9-9.4 mm in thickness, and four and a half-fold in level of mineralization (17.8X32.7% ash content). Holothuroids had the thickest body-wall tissues and contained the lowest levels of mineralization in their body-walls. Crinoids and ophiuroids had high levels of mineralization in their arms. Asteroid body-wall tissues varied the most in thickness and ash content (0.9-3.9 mm in thickness and 29.2-55.5% in ash content). Body-wall tissues of 19 species of echinoderms were tested for their feeding deterrent properties against the marine fishes La&on rhomboides (Linnaeus) and Cyprinodon variegatus (Lacepede), as well as the decapod crustacean Libinia emarginata (Leach). Equivalent sized pieces of fresh body-wall tissue of 16 species of echinoderms caused observable feeding deterrence responses in at least two of the three model predators. There was no significant correlation between body-wall thickness or percent ash and its palatability to any of the three model predators. Agar pellets containing ethanolic body-wall extracts of 12 of 18 echinoderm species caused observable feeding deterrence responses in the fish L. rhomboides. In similar experiments with the arrow crab Stenorhyncus seticornis (Herbst), using carrageenan fish-meal blocks as food models, no differences in consumption of control fish-meal and experimental body-wall extract blocks were detected. Our findings indicate that invertebrate and vertebrate predators may respond quite differently to echinoderm body-wall extracts. 01997 Elsevier Science B.V.

Keywords: Feeding deterrence; Echinoderms; Chemical defense; Structural defense

*Corresponding author. Tel.: + 852 2358 7310; fax: + 852 2358 1559; e-mail: pbryan@usthk.ust.hk

0022.0981/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII SOO22-098 1(96)02677-9

174 P.J. Bryan ef al. I .I. Exp. Mar. Biol. Ecol. 210 (1997) 173-186

1. Introduction

Echinoderms have a number of predators, including crustaceans (McLaughlin and Hebard, 1961; Aldrich, 1976), gastropods (Hughes and Hughes, 1971, 1981; Kabat, 1990), polychaetes (Glynn, 1984) and fish (Ormond et al., 1973; Wilson et al., 1974; Glynn, 1984) as well as other echinoderms (Jangoux and Lawrence, 1982). Although echinoderms are susceptible to predation, their abundance suggests that they are able to resist predators through effective defense mechanisms. Body-wall tissues of asteroids, crinoids, echinoids and ophiuroids generally contain high ash contents (50-90%) indicative of their heavy calcification (Lawrence and Guille, 1982), while most holothuroids often have more fleshy body-wall tissues and, therefore, lower ash contents. Nonetheless, body-wall tissues may contain 3-12 kJ/g dry tissue, with this energy predominantly attributable to relatively high levels of soluble and insoluble protein (McClintock et al., 1990). Additionally, echinoderms possess catch connective tissue, which allows them to alter the rigidity of their body-wall tissues (Motokawa, 1984a). This tissue may be important to the defense of echinoderms against predators by enabling tissues to quickly become rigid to provide structural support, or very soft to autotomize body parts before escape (Aldrich, 1976; Motokawa, 1984b). Once a predator penetrates the body-wall of an echinoderm, an even richer source of nutrients is available in the form of gonads (seasonally) and digestive tissues (Lawrence and Guille, 1982).

The most apparent defensive characteristic of echinoderms is often their tough and spiny outer body-wall, composed of high levels of embedded calcium and magnesium carbonate ossicles (Hyman, 1955). However, this generalization does not apply to all species of echinoderms. Holothuroids possess relatively minute ossicles interspersed throughout their body-wall tissues. The arms of crinoids and ophiuroids are heavily mineralized (Hyman, 1955) yet even these arms are palatable to some predators. Aronson (1988) found that five species of ophiuroids in the Bahamas are preyed upon by several species of fish.

Pedicellariae are pinching structures found in echinoderms which have been character- ized to serve a defensive role. However, pedicellariae are found only in asteroids and echinoids. Of the asteroids, pedicellariae are known to be present in the Orders Forcipulatida, Spinulosida and Valvatida (Hyman, 1955; Blake, 1987). The role of pedicellariae for defense in echinoderms must be viewed on the species level and not thought of as a generalized defense mechanism. Some pedicellariae are large and release toxins into their unfortunate recipients, whereas others are microscopic structures which may be too small to be effective deterrents against large mobile predators (Campbell, 1983).

Chemical defenses (Bakus, 1968; Bakus and Green, 1974; Lucas et al., 1979; Rideout et al., 1979) may complement morphological defenses in echinoderms and other marine invertebrates (reviewed by Bakus et al., 1986; Paul, 1992; Pawlik, 1993 and see Chanas and Pawlik, 1995). A number of studies have focused on the secondary metabolite chemistry of echinoderm tissues (e.g. Minale et al., 1982; Bumell and ApSimon, 1983; Stonik and Elyakov, 1988; Habermehl and Krebs, 1990). These studies have revealed that mid-polar saponin and saponin-like compounds are common in asteroid and

P.J. Bryan et al. I J. Exp. Mar. Bid. Ed. 210 (1997) 173-186 175

holothuroid tissues. The saponins of asteroids are more commonly steroidal glycosides, whereas holothuroid saponins are frequently derived from triterpenoids (Stonik and Elyakov, 1988). A few studies have also identified saponins in crinoid, ophiuroid and echinoid tissues (Ma&e et al., 1977); however, saponins are less common in these classes. Saponins have been shown to elicit detergent-like effects on cell membranes by interacting with membrane cholesterol to cause cell lysis (Seeman et al., 1973; Mackie et al., 1975). It is likely that saponins may function as feeding deterrents to predators of echinoderms.

The majority of studies that have addressed the bioactivity of echinoderm metabolites have screened compounds and crude extracts for their antifungal, antiviral, anti- inflammatory and antitumoral activity (Rinehart et al., 1981; Verbist, 1993). Previous studies have examined the toxicity of holothuroid and asteroid compounds or crude extracts to fish (Bakus, 1968; Mackie et al., 1975; McClintock, 1989) and marine invertebrate larval development (Ruggieri and Nigrelli, 1960). Only a few studies have addressed the role of body-wall tissues and crude ethanolic extracts of body-wall tissue compounds for ecologically relevant bioactivity. Lucas et al. (1979) demonstrated that saponins act as feeding deterrent agents in larvae of the sea star Acanthaster planci. Rideout et al. (1979) described feeding deterrent polyketide sulfates from several species of crinoids and Iorizzi et al. (1995) determined that several saponins and polyhydroxy steroids from the sea star Luidia clathruta inhibited bacterial growth and settlement of barnacle larvae.

The present study examines morphological (body-wall thickness and mineralization) and chemical (extracts containing mid-polar compounds) deterrent properties of the body-wall tissues of a suite of echinoderms from the northern Gulf of Mexico. As the echinoderms investigated either lacked pedicellaria or possessed small numbers of extremely minute pedicellaria, these potentially deterrent structures were not considered. Similarly, as no echinoids were investigated, nor were any of the other representative species notably spinated, spination of the body wall was not considered.

2. Materials and methods

2.1. Sites and collections

Asteroids, ophiuroids, holothuroids, and a crinoid were collected using a 9.1 m otter trawl from two offshore sites A (30” 00’ N; 87” 45’ W) and B (29” 48’ N; 87” 15’ W) and using SCUBA from two near-shore sites C (30” 15’ N; 86” 45’W) and D (27” 30’ N; 84” 15’ W) in the northeastern Gulf of Mexico (Hopkins et al., 1991).

Echinoderms were transported back to the laboratory in coolers and maintained live in aquaria until they were dissected to be employed in feeding assays. Body-wall tissues for chemical extraction were frozen immediately after collection and maintained at - 20°C. Model predatory crabs were collected by trawl from the echinoderm collection sites. Model fish predators were collected by seine from St. Joseph’s Bay, Florida.

176 P.J. Bryan et al. / J. Exp. Mar. Biol. Ecol. 210 (1997) 173-186

2.2. Structural defenses

Characteristics of echinoderm body-wall tissues which may contribute to structural defensive properties that were examined in the present study included the degree of mineralization and body-wall thickness. The dry weight percent ash (an indirect measure of mineralization) in the aboral body-wall of asteroids and ophiuroids, body-wall of holothuroids, and arms of the crinoid species was determined by obtaining two samples of tissue (1 g wet wt) from five individuals. Samples of wet tissue were weighed, lyophilized, reweighed and then ashed in a muffle furnace at 500°C for 4 h, and reweighed to establish percent ash content. Body-wall thickness of each species was determined to the nearest 0.1 mm by measuring the thickness of two samples of body-wall tissue, and arms of the crinoid, from five adult individuals of each species utilizing a vernier calliper.

A Pearson correlation analysis was conducted to determine whether significant relationships existed between the percentage of ash and the relative thickness of echinoderm body-wall tissues. Additional correlation analyses were performed to evaluate the relationship between the palatability of body-wall tissues to each of the model predators with the percentage of ash or thickness of echinoderm body-wall tissues.

2.3. Body-wall extraction

Echinoderms were dissected and body-wall tissues were weighed, lyophilized and reweighed. Saponins, which may prove to be the most predominant bioactive com- pounds isolated from echinoderms, are characteristically mid-polar compounds (Mackie et al., 1975). To extract saponin and saponin-like compounds, the lyophilized tissue was extracted in aqueous ethanol (ethanol:water, 80:20, Yasumoto et al., 1966). Tissues were extracted by submerging aboral body-wall tissues of at least five individuals of each species in the solvent mixture at a ratio of 3: 1 (volume:wt) for 24 h at 21°C under gentle agitation on a shaker table. The solvent mixture was decanted and filtered through Whatman # 1 filter paper and dried in a rotary evaporator. Solvent mixture extracts were desalted by resolubilization in ethanol:methanol (80:20) to precipitate oceanic salts present from the initial extraction. Desalted extracts were dried under nitrogen and weighed. The natural concentration (% dry wt) of extract recovered from the body-wall was determined for each species. Desalted and dried extracts were stored at - 20°C and rehydrated to appropriate concentrations in sterile seawater (32 ppt) prior to use in bioassays. Three 1 ml samples of each extract were evaporated to dryness under a fume hood and the dry extract weighed to the nearest 0.001 g. This value was used in conjunction with the percent of mineralization assumed to be in the body-wall (as ash free dry wt). Wet to dry weight ratios were used to calculate the percent of crude extract within the body-wall tissue.

2.4. Feeding deterrence assays

Experimental pellets (4% Sigma Agar dissolved in deionized water and containing 1.5% ground dry krill) were prepared containing echinoderm body-wall extracts at

P.J. Bryan et al. I J. Exp. Mar. Biol. Ecol. 210 (1997) 173-186 177

Table 1 Structural characteristics and ethanolic extract levels of echinoderm body-wall tissues

Thickness(mm) Ash% dry wt Extract% dry wt

Holothuroidea Holothuria lentigenosa Holothuria thomasae Isostichopus badionotous Crinoidea Comactinia meridionalis Ophiuroidea Astrocyclis caecilia Astrophyton muricatum Astroporpa annulata Asteroidea Anthenoides piercei Astropecten articulatus Chaetaster nodosa Goniaster tessalatus Henricia downeyae Linkia nodosus Luidia clathrata Narcisia trigonaria Oreaster reticulatus Tamaria halperni Tethyaster grundis Tosia parva

4.2 19.2 2.87 4.1 17.8 3.26 9.4 23.6 2.98

2.5 82.7 2.58

2.6 74.4 2.52 2.7 66.4 3.21 3.6 74.6 2.24

1.4 30.1 3.54 1.8 48.8 2.39 0.9 42.1 1.76 1.7 44.7 2.28 0.9 45.1 3.34 1.4 29.2 2.7 1 1.4 33.4 2.46 1.3 30.5 2.24 3.9 53.4 2.71 2.5 34.9 2.25 3.8 39.3 2.88 1.7 55.5 2.28

The percent extract dry weight is based on the dry weight of the desalted extract divided by the dry weight of the extracted tissue.

concentrations of 3.0 and 0.75 mg/ml agar. The higher concentration of body-wall extract employed in the pellets falls near the mean level of extract found in the echinoderm body-wall tissues examined (Table 1; mean extract concentration = 0.027 g/g dry tissue weight or 2.7%). In order to ensure the food value of the pellets was similar to that of body-wall tissues, the concentration of krill powder employed in the pellets resulted in pellets containing 10.2% soluble protein, a level near the mid range of soluble protein values we found in asteroid and ophiuroid body-walls from the Gulf of Mexico (McClintock et al., 1990; range = 3.3- 18.1% dry weight). Control pellets were comprised of a 4% agar containing 1.5% dry krill powder. Carmine particles were added to colour both control and experimental pellets to assist observations. Pellets were formed by inserting the tip of a glass pipette into agar to form a cylindrical pellet (I mm diam; 5 mm length).

Laboratory feeding trials for both fresh body-wall tissue and pellet assays consisted of introducing a piece of tissue or a pellet into one of ten 15gallon aquariums and observing the feeding responses of model predators (the sheepshead killifish Cyprinodon variegatus (Lacepede), the pinfish Lugodon rhomboides (Linnaeus), and the spider crab Libinia emurginatu (Leach), over a 1-min period. The pellet was released underwater by gently forcing air through the pipette. Pinfish and killifish were conditioned to feed on control pellets for 1 week prior to experimentation (McClintock and Vernon, 1990). As both species of fish showed a tendency to feed more readily when grouped, groups of

178 P.J. Bryan et al. I J. Exp. Mar. Biol. Ecol. 210 (1997) 173-186

five Cyprinodon variegatus or kgodon rhomboides were used. Crabs were tested individually. Feeding behaviours were evaluated by recording whether each tissue or pellet was ultimately accepted (ingested) or rejected by the subject fish or crabs over a I-min period. Twenty experimental tissues (measuring 3 mm on a side and varying in thickness) or experimental pellets and 20 control tissues (similar sized fish tissue) or control pellets were presented in a haphazard sequence to a group of fish in one of the experimental tanks for each echinoderm species body-wall or extract. The order of tanks in which pellets were offered was predetermined using a random numbers table. A Fisher’s Exact test (a = 0.05) was utilized to compare the consumption of control and experimental tissues or pellets (Sokal and Rohlf, 198 1).

The model predators described above included both echinoderm and non-echinoderm generalist predators. The sheepshead killifish Cyprinodon variegatus is allopatric with respect to the echinoderms tested and is unlikely to consume echinoderms as part of its diet. The pinfish L. rhomboides may be sympatric with several of the echinoderms tested and is known to include echinoderms in its diet (Walls, 1975). Both fish are generalist feeders (Weinstein et al., 1982; Perschbacher and Strawn, 1986; Luczkovich, 1988). The spider crab L. emarginata also occurs in sympatry with several of the echinoderms examined and is an echinoderm predator (Aldrich, 1976).

In the laboratory, individuals of the arrow crab Stenorhyncus seticornis (Herbst) were observed preying on the ophiuroid Ophioderma sp. and the asteroid Echinaster modestus (Perrier) (Bryan, pers. obs.). To broaden our analysis to yet another species that occurs in sympatry with the echinoderms investigated, and documented to feed on echinoderms in the laboratory, an additional feeding deterrent experiment was performed. Fifteen S. seticornis were maintained in laboratory aquaria in individual compartments. Assays with this crab were conducted using carrageenan blocks as food models. To prepare experimental blocks, echinoderm body-wall extracts were mixed with a 2% carrageenan containing a 4% fish meal to yield a final concentration of 3.0 mg extract/ml carrageenan. The fish-meal treated carrageenan blocks contained 22.3% soluble protein (dry wt), a level similar to the highest levels of soluble protein measured in asteroid and ophiuroid body-wall tissues from the Gulf of Mexico (McClintock et al., 1990). Therefore, this provided a conservative measure of the chemical feeding deterrent properties of echinoderm body-wall extracts. Control blocks were comprised of a 2% carageenan containing only a 4% fish meal. Heated solubilized carageenan was allowed to cool to 40°C before the body-wall extracts were added. The carageenan was then poured into small petri dishes (5 cm diameter X 1 cm deep) and allowed to solidify. Blocks measuring 1 cm3 were cut from the carrageenan and weighed. An experimental block containing fish meal and echinoderm body-wall extract along with a control block containing only fish meal were slid onto a plastic cable tie (5 X 0.5 cm). One end of the cable tie was marked with indelible ink to record the position of the control block. The two pre-weighed food blocks were presented to an individual S. seticornis, which was subsequently allowed to feed for a period of 12 h. The blocks were then retrieved and re-weighed. A second group of control blocks was weighed and placed in similar fashion in seawater for a 12 h period and then reweighed to permit a measurement of weight gain by way of absorbed water under the experimental test conditions. Differential weight gain by absorption of water was factored into the final determination of the actual

P.J. Bryan et al. I J. Exp. Mar. Biol. Ecol. 2/O (1997) 173-186 179

amount of weight of the food consumed by each crab. The weights of the control and experimental food blocks were compared using a Student’st-test (a = 0.05) (Sokal and Rohlf, 1981).

3. Results

The percent ash (mineralization) and body-wall thicknesses of the echinoderms investigated are presented in Table 1. There was no significant correlation between the thickness of the body-wall and the percentage of ash of the echinoderm body-wall tissues tested; the Pearson correlation value (PCV) was 0.139 and p = 0.56. Moreover, there was no correlation between body-wall thickness or percent ash of body-wall tissues and their palatability to the fishes Lagodon rhomboides (PCV = 0.054, p = 0.827; PCV= - 0.297, p = 0.218), Cyprinodon variegatus (PCV= 0.231, p = 0.341; PCV = - 0.461, p = 0.057), or the spider crab Libinia emarginutu (PCV= 0.151, p = 0.537; PCV = - 0.119, p = 0.629), respectively.

The killifish Cyprinodon vuriegutus demonstrated distinct rejection of the fresh body-wall tissues of 18 of the 19 echinoderms tested (Table 2). When tested, fresh body-wall tissues from 16 of 19 echinoderms studied were rejected more frequently than controls by the pinfish Lagodon rhomboides (Table 2). Only fresh body-wall tissues of the asteroids Tumuriu hulperni and Henriciu downeyue were rejected more often than controls by the spider crab Libiniu emarginutu (Table 2).

The feeding deterrent activities of ethanolic echinoderm body-wall extracts embedded in artificial food pellets or blocks and presented to the pinfish Lugodon rhomboides and the arrow crab Stenorhyncus seticornis are presented, respectively, in Tables 3 and 4. Lagodon rhomboides significantly rejected agar pellets containing 3.0 mg body-wall extract/ml agar from 12 of 18 echinoderm species. When tested at a concentration of 0.75 mg/ml agar, only pellets containing the body-wall extracts of the basket star Astrocyclus caeciliu and the asteroid Tosia purvu were rejected more often than controls (Table 3). When tested at a concentration of 3.0 mg/ml carrageenan, echinoderm body-wall extracts had no feeding deterrent effects on the arrow crab S. seticornis (Table

4).

4. Discussion

Structural characteristics of echinoderms including the degree of mineralization and body-wall thickness have been shown to be effective deterrents to some generalist predators (Wilson et al., 1974; Bingham and Braithwaite, 1986). However, other generalist predators such as balistid, labrid and sparid fishes have mouth parts that accommodate the consumption of heavily calcified tissues (Shupp and Paul, 1994; Chanas and Pawlik, 1995). Echinoderm defensive attributes also include autotomization of body parts, a likely strategy to withstand sub-lethal predation (Emson and Wilkie, 1980). In the present study we found no significant correlation between the consumption of pieces of echinoderm body-wall tissues offered to model predators in laboratory assay

180 P.J. Bryan et al. I J. Exp. Mar. Biol. Ecol. 210 (1997) 173-186

Table 2 Feeding deterrence of echinoderm body-wall tissues

Lagodonrhomboides Cyprinodonvariegatus Libiniaemarginata

Holothuroidea Holothuria lentigenosa Holothuria thomasae Isostichopus badionotous Crinoidea Comactinia meridionalis Ophiuroidea Astrocyclis caecilia Astrophyton muricatum Astropotpa annulata Asteroidea Anthenoides piercei Astropecten articulatus Chaetaster nodosa Goniaster tessalatus Henricia downeyae Linkia nodosus Luidia clathrata Narcisia trigonaria Oreaster reticulatus Tamaria halperni Tethyaster grandis Tosia parva

0.0016* 0.1864

<0.0001*

< 0.0001*

0.0035* 0.0007*

< 0.0001*

0.0563 0.0003* 0.577 1

< 0.0001* 0.0035* 0.0016* 0.3299 0.0003* 0.0035*

<0.0001* 0.3299 0.0035*

< 0.0001* 0.5771

< 0.0001*

< 0.0001* 0.3299

0.0075* 0.9999 < 0.0001* 0.9999 <0.0001* 0.1864

0.0003* 0.9999 < 0.0001* 0.5771

0.0075* 0.5771 <0.0001* 0.1036 < 0.0001* 0.0001*

*0.0016 0.3299 0.0016* 0.9999

<0.0001* 0.9999 0.0003* 0.9999

< 0.0001* 0.0075* < 0.0001* 0.9999 < 0.0001* 0.3299

0.9999 0.5771 0.9999

p-values are presented for the assay of each echinoderm tissue fed to a model predator along with control tissues. Statistically significant feeding deterrence (p < 0.01 from a Fisher’s Exact test) is indicated with an asterisk. The three model predators greatly differed in their consumption of echinoderm body-wall tissue. Only three of the species showed no deterrent effect to any of the model predators. The tissues from Tamaria halperni and Henricia downeyae significantly deterred feeding of all three model predators.

and either body-wall thickness or mineralization. It is evident that neither of these factors is directly responsible for determining palatability of the echinoderm body-wall tissues examined in our study. Under natural conditions, intact tissues may be more difficult to consume than the body-wall pieces we used in our feeding trials. Conclusions about the ultimate role of structural defenses in echinoderm body-wall tissues would be facilitated by experimental studies which load body-wall skeletal elements (ossicles) into model food pellets containing naturally occurring concentrations of extracts and test them against ecologically relevant predators. Nonetheless, our observations indicate that factors other than body-wall thickness and degree of mineralization, such as chemical deterrents, are important in determining palatability.

Specialist predators are likely to have co-evolved with their prey and developed mechanisms to overcome structural or chemical defenses (reviewed in Paul, 1992). These defenses are then directed towards more generalist predators who sample many potential food sources and learn which ones to continue eating and which ones to avoid (Paul et al., 1990; Paul and Pennings, 1991; Rogers and Paul, 1991; Vrolijk et al., 1994). We found that two generalist fish predators, the pinfish Lagodon rhomboides (Stoner,

P.J. Bryan et al. I J. Exp. Mar. Biol. Ecol. 210 (1997) 173-186 181

Table 3 Feeding deterrence of echinoderm body-wall extracts to the Pinfish Lagodon rhomboides

Percent of pellets rejected

3.0 mg/ml 0.75 mglml Control

Holothuroidea Holothuria lentigenosa 100* 20 0 Isostichopus badionotous 10 0 0 Crinoidea Comactinia meridionalis Ophiuroidea A.strocyclis caecilia Astrophyton muricatum Astroporpa annulata Asteroidea Anthenoides piercei Astropecten articulatus Chaetaster nodosa Goniaster tessalatus Henricia downeyae Linkia nodosus Luidia clathrata Narcisia trigonaria Oreaster reticulatus Tamaria halperni Tethyaster grandis Tosia parva

0

87% 67*

80*

27 20 87* 47 56* 40 72% 52* 70* 40

100* 30 25 30 64* 27 46 27 60* 44 20 20 90* 70*

0

73* 13 13

0

0 0 0

0 0.5 0

0

0

0 0 0

0 0 0

The effect of ethanolic extracts on the pinfish was assayed at two concentrations. Pellets were offered to groups of fish in a haphazard sequence and observed for a period of 1 min to determine acceptance or rejection of the pellet,

1980) and the killifish Cyprinodon variegatus (Perschbacher and Strawn, 1986), rarely accepted echinoderm body-wall tissues. Cyprinodon variegatus rejected all echinoderm body-wall tissues with the exception of the body-wall tissues of the holothuroid Holothuria thomasue; this species has a soft, poorly mineralized (18% ash) body-wall which is likely to afford little structural defense. This suggests a trade-off between this aspect of structural defense and chemical defense. The predatory crabs tested, also feeding generalists (Bryan, pers. obs.), were almost completely undeterred by the compounds that deterred both species of fish. Therefore, feeding deterrent responses can vary dramatically across predators (Hay et al., 1988; Wylie and Paul, 1989). Moreover, the differences in deterrence responses of generalist invertebrate and vertebrate predators confound generalizations relating feeding strategies to the evolution of chemical defense.

The use of standardized food models containing echinoderm body-wall extracts facilitates an evaluation of the role of chemical deterrents in mediating tissue palatability across echinoderm classes. Among the Holothuroidea, extracts of the body-wall tissues of both Isostichopus budionotus and Holothuriu lentigenosu caused feeding deterrence in fish. In contrast, we found that the arrow crab Stenorhyncus seticornis was capable of ingesting these body-wall extracts. Chemical activity has been detected in a variety of temperate and tropical holothuroids (Bakus, 1968; Bakus and Green, 1974). These

182 P.J. Bryan et al. / J. Exp. Mar. Biol. Ecol. 210 (1997) 173-186

Table 4 Palatability of echinoderm body-wall extracts to the Arrow crab Srenorhyncus sericornis

Mean Percent Consumed

Control Experimental

Holothuroidea Holothuria lentigenosa 82.1 83.4 Isosrichopus badionotous 12.6 81.8 Crinoidea Comactinia meridionalis 76.8 79.6 Ophiuroidea Astrocyclis caeciliu 74.3 81.3 Astrophyton muricatum 81.1 78.9 Astroporpa annulara 88.4 85.4 Asteroidea Anthenoides piercei 74.3 71.3 Astropecten arriculatus 91.5 87.7 Chaetasrer nodosa 78.7 77.8 Goniaster ressalatus 17.2 79.1 Henricia downeyae 77.4 81.3 Linkia nodosus 68.9 13.4 Luidia clathrara 71.8 79.2 Narcisia trigonaria 88.4 87.3 Oreasrer reticulatus 74.3 77.6 Tarnariu halperni 73.1 71.2 Tethyuster grandis 85.2 79.6 Tosia parva 91.2 93.1

The effect of ethanolic extracts on consumption of carrageenan cubes by S. seticornis was assayed. Control and experimental cubes were weighed before and after being presented to crabs for a 12 h period. Percent values of the amount of food cubes eaten are presented below. A Student’s r-test was utilized to compare the mean (n = 15) amount of cubes consumed between controls and experimentals for each echinoderm extract.

studies measured toxicity in fish exposed to holothuroid extracts placed in seawater; subsequent investigations have shown that there is not necessarily a correlation between toxicity and feeding deterrence in marine invertebrates (Pawlik et al., 1995). Holothuroids generally have soft body-wall tissues and are exposed to predators while foraging (Bakus, 1968). Nonetheless, they appear to have few predators (Bakus, 1968, 1981), indicating the potential importance of chemical defense. Chemical investigations of holothuroid tissues have revealed a suite of novel saponins, many of which have demonstrated cytotoxicity (Stonik and Elyakov, 1988).

As body tissue extracts of the comatulid crinoid Comactinia merdionalis were not rejected by any of the model predators, there is no evidence that mid-polar compounds cause deterrence. The rejection of whole pieces of body tissues by both species of fish indicates either structural defenses, or defense attributable to chemical deterrent compounds of high or low polarity. The level of calcification was particularly high in the arms of C. meridionalis (82.7%) and likely accounts for its unpalatability. Rideout et al. (1979) detected polyketide sulfates in 5 of 20 species of coma&did crinoids and demonstrated that these may act as fish feeding deterrents.

The body-wall extracts of all three gorgonocephalid ophiuroids were deterrent to the

P.J. Bryan et al. I J. Exp. Mar. Bid. Ed. 210 (1997) 173-186 183

pinfish Lagodon rhomboides. The palatability of ophiuroids has been investigated by Hendler (1984) and Aronson (1988). Both studies found that fish discriminated in their feeding when presented a suite of ophiuroid species, with some species selected over others. Neither study examined whether deterrence was structurally or chemically based. Our experimental results indicate that ophiuroids may harbour defensive chemistry. The secondary metabolite chemistry of ophiuroids has been investigated (Ma&e et al., 1977; Riccio et al., 1985; D’Auria et al., 1991). Although a very low incidence of the typical saponins or steroidal glycosides were detected, several unusual sulfates were found that may have deterrent properties.

By far the most representative class of echinoderms examined for defensive chemistry in the present study were members of the Asteroidea. We found that 10 of 12 (83%) of the body-wall extracts from asteroids were deterrent against the pinfish Lugodon rhomboides. In contrast, body-wall tissues and body-wall extracts were not deterrent against the spider crab Libinia emarginata and the arrow crab Stenorhyncus seticornis. This indicates that different model predators show differential feeding responses to asteroid body-wall tissues, as seen with representatives of the other echinoderm classes we examined. Moreover, it likely indicates an ability of both species of crabs to tolerate, sequester or deactivate defensive compounds (Wylie and Paul, 1989; Vrolijk et al., 1994). Similarly, Hay et al. (1988) found that amphipods are capable of feeding on algae that is defended with secondary metabolites. The secondary metabolite chemistry of two of the asteroids investigated in the present study has been investigated. Luidia clathrata elaborates saponins and polyhydroxy steroids that have been shown to have biological activity against microbes and prevent settlement in both barnacle and bryozoan larvae (Iorizzi et al., 1995). Henricia downeyae has been found to produce two novel sulphated steroid glucoronides (Downeyoside A and B) that are cytotoxic (Palagiano et al., 1995). These compounds may also act as feeding deterrents; in some cases having demonstra- tive membrane irritant qualities (Mackie et al., 1975).

Acknowledgments

We thank K. Marion, J. Lawrence, J. Gauthier and two anonymous reviewers for their helpful comments on this manuscript. We also wish to acknowledge the assistance of the captains and crews of the RV Suncoaster, RV Bellows, and RV Verrill. Logistical support was provided by Dauphin Island Sea Laboratory and the Alabama Marine Environmental Science Consortium. This work was supported by NSF EPSCoR grant No. EHR-9108761 to J.B.M, T.S.H., Ken Marion and Stephen Watts.

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