J. Exp. Mar. BioL Ecol., Vol. 147 (1991) 163-175 © 1991 Elsevier Science Publishers B.V. 0022-0981/91/$03.50

163

JEMBE 01563

Biochemical and energetic composition, population biology, and chemical defense of the antarctic ascidian

Cnemidocarpa verrucosa Lesson

James B. McClintock ~, John Heine 2, Marc Slattery ~ and James Weston 2 i Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA; 2Moss

Landing Marine Laboratories, Moss Landing, California, USA

(Received 27 July 1990; revision received 3 October 1990; accepted 16 November 1990)

Abstract: The biochemical and energetic composition, population biology (size-weight relationship, abundance and size-frequency distribution) and chemical defense of the antarctic ascidian Cnemidocarpa verrucosa Lesson was investigated at McMurdo Sound, Antarctica, during the austral summer of 1989. The organic content (% organic material, g- m dry tissue wt) of individual body components (tunic, body wall, endocarps, intestines, mature ovitestes and branchial basket) was generally high, with values ranging from 44.5% in the endocarps to 83.9% in the gonads. Most of this material was in the form of NaOH-soluble and insoluble protein. Tissue energy values ranged from 15.1 (tunic) to 22.4 (gonad) k J . g - ~ dry wt. Body height (cm) was positively related to body weight (g dry wt) by an exponential function. A representative individual (14cm height, 550g wet wt) contained a total of 493 kJ with most (75%) of this energy ~',tributable to the body wall and tunic. The gonadal index [(kJ ovitestes- total kJ - ! ) x 100] for sexually mature individuals collected in November was 17.2 _+ 4.7 (n = 6). Population densities of C. verrucosa at depths of 20-30 m were 0.4 ind. m - 2 at a site 3 km north of McMurdo Station. Energetic densities were estimated to be 197 kJ. m - 2. Size-frequency analysis revealed four modal peaks that probably represent distinct age cohorts, and may indicate predictable, annual recruitment events. Bioassays revealed that the tunic was noxious to sympatric pelagic and benthic fish, e,s well as an allopatric model fish. However, aqueous tunic extracts did not cause mortality in sea urchin sperm indicating the noxious compound(s) is not cytotoxic, pH values for body components ~ece weakly acidic or neutral (5.86-6.93). Mature ovitestes were rejected by sympatric pelagic fish suggesting that gametes may be chemically defended. Although this common antarctic ascidian represents a significant resot~rce of materials and energy, its tunic is tough and noxious, and probably provides an effective means of defense against potential predators.

Key words: Antarctica; Ascidian; Chemical defense; Energetics

INTRODUCTION

A number of investigators have postulated that due to low levels of predation by browsing fish, selection for chemical defense in sessile marine invertebrates at high latitudes should be low compared with temperate, and especially tropical, systems (Bakus, 1974, 1981; Bakus & Green, 1974; Green, 1974; Vermeij, 1978). Predation by browsing fish does decrease with increasing latitude (Neudecker, 1979; Palmer, 1979).

Correspondence address: J.B. McClintock, Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

164 J.B. McCLINTOCK ET AL.

Nonetheless, recent studies have shown that a variety of antarctic marine invertebrates representing at least five phyla are chemically defended (bryozoans: Winston & Bernheimer, 1986; sponges: Molinski & Faulkner, 1987; McClintock, 1987; echinoderms: McClintock, 1989a; cnidarians: McClintock et al., in press; molluscs: McClintock & Janssen, 1990). High levels of chemical defense among antarctic species may be related to an extremely stable environment (Pearse et al., 1991) of substantial geological age, which has facilitated benthic communities to be structured in large part by competition and predation (i.e., biologically accommodated; sensu Dayton et al., 1974).

Cnemidocarpa verrucosa Lesson is one of the most conspicuous and abundant antarctic ascidians occurring at shallow depths below the zone of ice scour (> 20 m) (Kott, 1969; Dayton et al., 1974). Its distribution is circumpolar in the antarctic and subantarctic and it has been recorded up to depths of 400 m (Kott, 1969). These large solitary ascidians are often found attached by their bases to cobble or boulders, some- times in clusters of several individuals (McClintock, pers. obs.). Although they may represent a significant trophic resource, the only potential predator observed crawling on the body surface is a chrome yellow lamellarian gastropod (probably Phyline

antarctica), which drills into the tunic to deposit its eggs (Dayton et al., 1974). A large suite of antarctic sea stars co-occur with C. verrucosa, many of which are known consumers of other sessile macroinvertebrates (e.g., sponges; Dayton etal., 1974; McClintock, 1987). However, although some sea stars feed on ascidians (Guitierrez & Lay, 1965; Gulliksen & Skaeveland, 1973; Parry, 1984), none of these potential preda- tors have been observed feeding on this ascidian in McMurdo Sound (Dayton et al., 1974; McClintock et al., pers. obs.). Moreover, no evidence exists which would suggest that sympatric benthic fish such as Trematomus bernacchii browse on C. verrucosa

(Moreno, 1980; Foster & Montgomery, in press). Studies of temperate and tropical ascidians have revealed that bioactive chemicals

are frequently sequestered in the outer tunic (Millar, 1971; Stoecker, 1980a). These chemicals are primarily alkaloids, amino acid-derived metabolites (Faulkner, 1984, 1986, 1987) or vanadium (Webb, 1939; Ciereszko et al., 1963; Swinehart et al., 1974; Danskin, 1978; Stoecker, 1980a,b,c). Moreover, some ascidians have specialized bladder cells in their body walls which secrete sulfuric acid that may play a role in defense (Webb, 1939; Stoecker, 1980a,b,c; but see Parry, 1984). Although the chemical activity of temperate and tropical ascidians has received much attention, no studies have been conducted with polar species.

One objective of this study was to examine the nutritional quality of C. verrucosa as a potential prey. By measuring the biochemical and energetic composition of the body components, it was also possible to consider their function and compare the relative allocation of materials and energy to different processes (e.g., somatic vs. reproductive output). Moreover, both energy content of an intact individual and energetic density of the population could be estimated. The sect~nd objective of this study was to provide the first quantitative assessment of the population abundance and size-frequency

CHEMICAL DEFENSE OF AN ANTARCTIC ASCIDIAN 165

distribution of C. verrucosa. Finally, this study extends the evaluation of chemical defense m¢chaiiisms in polar marine invertebrates to a~other phylum by determining whether apparent low levels of predation on C. verrucosa are related to the presence of noxious or toxic compounds.

MATERIALS AND METHODS

BIOCHEMICAL AND ENERGETIC ANALYSES

C. verrucosa (n = 10) of varying sizes were collected by hand using scuba ~., November 1989 from hard-bottom substrata 20-30 m below the sea ice near Arrival Heights, McMurdo Sound, Antarctica (77 ° 34' S, 164 ° 39' E). Individuals were measured (height) and weighed (wet wt) prior to dissection into the tunic, body wall, endocarps, intestines, ovitestes (if present) and branchial chamber. The tunic was cleaned of any fouling organisms. Each body component was weighed, subsampled, lyophilized and ground into a fine powder in a Wiley Mill. Ash was determined by placing tissues into a muffle furnace for 4 h at 500 ° C. Methanol-chloroform soluble lipid was measured gravimetrically using the technique of Freeman et al. (1957). TCA- soluble carbohydrate and NaOH-soluble protein were measured colorimetrically using the techniques of Dubois et al. (1953) and Bradford (1976), respectively. Insoluble protein was calculated by subtraction (Lawrence, 1973).

Energetic composition ( k J . g - l dry wt) of each body component was calculated indirectly by multiplying the dry wt of each tissue sample by the level of each organic class and its energetic equivalent (Brody, 1945). The energetic content (total k J . g - 1

dry wt) of each tissue was then multiplied by the total dry wt of each body component, and the totals for each component summed to yield the energy content of an intact representative individual. A gonadal index was calculated for six reproductively mature adults as the kJ ovitestes" total kJ - l x 100. Reproductive maturity was ascertained by examining gamete smears under a compound microscope. Moreover, several individuals spawned gametes while being held in the laboratory reflecting the mature reproductive condition of the population.

POPULATION BIOLOGY

A length-weight regression was calculated using the individuals collected for the biochemical and energetic analyses. The abundance and size-frequency distribution of C. verrucosa was determined for a population at Compromise Cove, ~ 3 km north of McMurdo Station. Numbers and height of individuals were measured in situ by divers swimming along the length of eight 25-m transects running parallel to one another along a depth gradient from 20-30 m. Each diver carried a section of PVC pipe to facilitate counting individuals within 1 m of the transect line. The energetic density (kJ" m- 2) of this population was estimated by calculating the dry wt equivalent energy content of the entire population and dividing by the number of square meters sampled.

166 J.B. McCLINTOCK ET AL.

ICHTHYONOXICITY, CYTOTOXICITY AND PH ASSAYS

The somatic body components (tunic, body wall, endocarps, intestine, branchial basket) of C. verrucosa were cut into cubes (1 x 1 x 1 cm) and offered to 10 different individuals of each of two species of antarctic fish with different foraging habitats (Pagothenia borchgrevinki:generally a pelagic forager which feeds occasionally on benthic organisms; T. bernacchii: exclusively a benthic forager). Pieces (1 x 1 x ~ cm) ofmature gonads (ovitestes) also were presented to 10 P. borchgrevinki. Gonad bioassays were restricted to this fish species since planktonic embryos would only be encountered by pelagic fish. 10 control trials were conducted with both species of fish, each trial consisting of" presenting individual fi~h with 1 x 1 x 1-cm pieces of'tail mu=de from the anta~r:tic cod Dissostichus mawsoni. The order of presentation of the control or experi- medical tissue was randomized. Ingestion or rejection (mouthed and then spit out) of both experimental (ascidian) and control (cod) pieces was recorded.

In order to discriminate whether fish may reject tunic tissues on the basis of their tough consistency or noxious chemicals, tunic material was lyophilized and ground into a fine powder using a Wiley Mill. This material was embedded at a concentration of 5 9o (g" ml- t ) in gum agar containing a 5 % fish meal, and cylindrical agar pellets (1 mm diameter, 4 mm height) were prepared. Control agar pellets contained only 5 ~o dry fish meal. Killifish Fundulus grandis collected from shallow estuarine waters near Dauphin Island, Alabama, were used as model predators. A group of'five fish were presented with either an experimental or control pellet, since preliminary experiments had indicated that fi~h fed more readily when in a small group. The order of presentation of the pellets was randomized. A total of 37 pellets (19 experimental, 18 control) were presented to fish and pellet ingestion or rejection recorded.

Aqueous tunic extract was prepared by homogenizing a known wet wt of tunic with an equal volume of" cold seawater and filtering out particulate material. Cytotoxicity assays were conducted using the mature spermatozoa of the antarctic sea urchin Sterechinus neumeyeri. Gametes were collected by intracoelomic injection of KCI. A 100-#1 aliquot of fresh dry sperm was placed on spotting plates maintained at 0 o C and 250 #1 o[ aqueous tunic extracts diluted to 3, 0.3, 0.03 and 0.003~ were added (n = 10 spots, concentration of extract - ~ ). The activity of sperm was scored positive (> 75 ~o active) or negative (< 75% active) after a 20-min incubation period. 10 control sperm assays were conducted simultaneously using seawater alone. In both the ichthyonoxicity and cytotoxicity assays statistical significance was determined using a Fisher's exact test.

Three individual ascidians were dissected into the tunic, body wall, endocarps, intestines, ovitestes and branchial basket and each tissue was homogenized. The pH of the homogenates then was measured using an Orion pH meter (SA250).

C H E M I C A L DEFENSE OF AN ANTARCTIC A S C I D I A N 167

R E S U L T S

BIOCHEMICAL A N D ENERGETIC C O M P O S I T I O N

The biochemical composition of somatic and reproductive body components varied greatly in C. verrucosa (Table I). The gross organic contents of the somatic tissues

T'B!.E I

Biochemical composition (~o dry wt) of body components ofC. v e r r u c o s a from McMurdo Sound, Antarctica (~ + 1 SE).

Body n Ash Carbohydrate Lipid Soluble Insoluble component protein protein

--9

Tunic 10 38.8 + 1.7 0.7 + 0.05 4.9 + 0.9 6.2 + 0.5 49.1 + 2.0 Body wall 10 27.3 + 1.4 0.8 + 0.06 10.7 + 2.9 18.0 + 1.0 43.1 + 3.3 Endocarps 6 55.5 + 2.3 0.5 + 0.07 6.2 + 1.3 5.9 + 1.0 30.6 + 2.3 Intestines 7 25.9 + 0.6 1.3 + 0.75 11.6 + 0.8 11.4 + 0.8 49.8 + 1.2 Ovitestes 6 16.1 + 0.6 1.1 + 0.13 16.1 + 2.1 14.1 + 1.3 51.3 + 2.4 Branchial 8 36.8 + 0.7 0.7 + 0.04 8.0 + 0.6 12.0 + 0.9 41.9 + 2.4

basket

ranged from 44.5 to 74.1 ~o dry wt in the endocmps and intestines, respectively. The ovitestes were highest in organic material (83.9~/0 dry wt), with most of this material attributable to high levels of lipid and soluble and insoluble protein. Levels of soluble carbohydrate were generally low in all body components (0.5-1.3 ~ dr~ wt). Lipid levels in somatic tissues ranged from low values of 4.9-6.2 ~ dry wt in the tough outer tunic and endocarps to levels 2-3 times higher in the body wall, intestines and branchial basket (11.4-18.0~/o dry wt). Collectively, protein (seluble and insoluble)made up the single most prevalent organic constituent in all body tissues, with values ranging from 36.5 o~ dry wt in the endocarps to 65.4°" 0 dry wt in the ovitestes.

/ O

The energetic composition of the somatic and reproductive tissues was also variable (Table II). The endocarps clearly had the lowest energy content (9.5 kJ "g- l dry wt) of any of the body components, with particularly low energy values associated with lipid and soluble carbohydrate. Levels of energy in the tunic and body wall were twice as high, with values of 15.2 and 17.7 kJ .g-1 dry wt, respectively. Most of this energy was

- !

attributable to the insoluble protein. The ovitestes were highest in energy (22.6 kJ .g dry wt), with comparatively high levels of lipid-derived energy (6.9 k J ' g - l dry wt). A representative C. verrucosa weighing 550 g wet wt and with a height of 14 cm contained a total of 493.4 kJ (tunic" 217.7; body wall" 150.5; endocarps" 8.8; intestines" 49.5; ovitestes: 47.4; branchial basket: 19.5). The gonad index [(kJ ripe ovitestes.totat kJ - l) x 100] ofsexually mature individuals was 17.2 + 4.7 (2 + 1 SD;n = 6 individuals ranging from 6.9-14.4 cm height and 125-552 g wet wt). Individuals < 60 g wet wt had very small ovitestes or lacked them completely.

168 J.B. McCLINTOCK ET AL.

POPULATION BIOLOGY

The relationship between body height and dry wt was significantly correlated (r2= 0.81, P < 0 . 0 5 ) , and was best described by the exponential equation y = 0.6616 + 10 °'13162x (Fig. 1). C. verrucosa were found to occur at an overall density

TABLE II

Energetic composition ( k J . g - t dry wt) of body components of C. verrucosa from McMurdo Sound, Antarctica (~ _+ 1 SE).

Body component n Carbohydrate Lipid Soluble Insoluble Total protein protein

Tunic 10 0.12 + 0.01 2.0 + 0.3 1.5 + 0.1 11.6 + 0.5 15.22 Body wall 10 0.14 + 0.05 3.3 + 0.3 4.1 + 0.3 10.2 + 0.8 17.74 Endocarps 6 0.03 + 0.09 0.9 + 0.3 1.3 + 0.3 7.3 + 0.6 9.53 Intestines 7 0.22 + 0.02 4.7 + 0.4 2.7 + 0.2 12.0 + 0.4 19.62 Ovitestes 6 0.20 + 0.02 6.9 + 1.0 3.4 + 0.4 12.1 + 0.6 22.60 Branchial basket 8 0.12 + 0.01 3.2 + 0.2 2.8 + 0,2 10.2 + 0.7 16.32

A

>, l , , .

40-

W

~ a ! i | ! ! |

0 2 4 6 8 10 12 14

• !

1 6

Height (cm) Fig. !. Relationship between total dry body weight (g) and body height (cm) for C. verrucosa up to a height of 16 cm. An exponential equation O' = 0.6616 + 10 ° ' i3162x, r 2 = 0.81, n = 10) best describes line. Note that

there were two individuals ~ 6.5 cm height and these points overlap.

of 0.4 ind. m - 2 at the Compromise Cove study site (20-30 m depth). Although no rigorous analysis of patterns of distribution was conducted, individuals were often found clumped in patches of up to 10 individuals, attached to the surfaces of cobbles or large boulders. On the basis of this population density, energetic population levels were estimated to be 197 kJ. m - 2. This is a conservative estimate of population energy level, as small individuals ( < 10 cm height) frequently lacked mature ovitestes. Size-fre-

CHEMICAL DEFENSE OF AN ANTARCTIC ASCIDIAN 169

quency analyses of 89 individuals revealed a population characterized by at least four distinct modal peaks (Fig. 2).

ICHTHYONOXICITY. CYTOTOXICITY AND PH ASSAYS

The sympatric fish T. bernacchii and P. borchgrevinki demonstrated significant (P < 0.001) rejection of pieces of fresh tunic (Table III). P. borchgrevinki also showed

12

A

v

> , (.1 c 0

O" Q L .

U.

Cnemidocarpa verrucosa lO

8

6

4

jlj, uJll u,,HI IH, , 0 • • . . . . • , • • , • • • • • • • • • • . . . . . .

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 5 2 6 2 8

Height (cm)

Fig. 2. Size-frequency distribution (o,, = jo) of C. verrucosa (n 89) at Compromise Cove, 3 km north of McMurdo Station. Individuals included in population census occurred between 20-30 m depth.

TABLE III

Ichthyonoxicity of body components of C. verrucosa from McMurdo Sound, Antarctica. Shown is percen- tage of body component pieces rejected by sympatric fish (n = 10). Controls (n = 10. fish species-~), consisting of pieces of tail muscle ofD. mawsoni, were always ingested. * Significant at 0.001 level (Fisher's

exact test); - , not tested.

F:sh Body component

Tunic Body wall Endocarps Intestines Ovitestes Branchial basket

100" 0 40 0 - 40 T. bernacchii (benthic)

P. borchgrevinki (pelagic)

100" 0 100" 0 100" 100"

significant (P < 0.0001) rejection of endocarps, ovitestes and the branchial basket. The allopatric fish F. grandis exhibited significant (P < 0.05) rejection oflyophilized, ground tunic embedded in agar fish-meal pellets (tunic pellets with fish meal: 18 rejected, one accepted; control pellets with fish meal alone: six rejected, 12 accepted). No significant

170 J.B. MeCLINTOCK ET AL.

levels of sea urchin sperm cytotoxicity were detected in controls or at any of the concentrations of aqueous tunic extract tested.

The pH (7 + 1 SD, n = 3) of the homogenized tunic (6.93 + 0.05), body wall (6.46 + 0.04), endOcarps (6.47 + 0.07), and intestines (6.61 + 0.03) was close to neu- tral. The ovitestes (5.86 + 0.07) and branchial basket (6.05 + 0.05) were slightly more acidic.

DISCUSSION

The organic content (% organic material.g-~ dry wt)of the body components of C. verrucosa are, with the exception ofthe endocarps, relatively high and similar to other cold water ascidians (Wacasey & Atkinson, 1987). Most of the organic material is in the form of lipid and protein, particularly insoluble protein, which is probably structural in nature (e.g. components of connective tissues). Not surprisingly, the tough outer tunic was higher in insoluble protein than the softer tissues of the internal body wall. As insoluble protein was calculated by subtraction, it is possible that some fraction of the material considered to belong in this "insoluble" category is a protein-polysaccharide complex as seen in a number of ascidians including the tunic of Halocynthia aurantium

(Smith & Dehnel, 1970). The high levels of lipid (10.7% dry wt) and soluble protein (18.0%) found in the body wall tissues indicate that this body component may serve as a nutrient storage organ as in some other marine invertebrates (McClintock, 1989b; Saito & Watts, 1989).

The ovitestes contained the highest levels of lipid of any of the body components; most of this material is p~ ~ ~umably attributable to developing or mature oocytes. High amounts of"insoluble protein' in the ovitestes may be somewhat inflated due to copious nucleic acid material in the testicular fraction of the ovitestes. As insoluble protein is calculated by subtraction, nucleic acids, normally at very low levels, are included in this category. The endocarps contained particularly low levels of organic material, and were characterized by high amounts of ash and reduced levels of carbohydrate, lipid and protein. The functional significance of the endocarps in this species, and a number of other ascidians, is unknown (C. Young, pers. comm.). In C. verrucosa the endocarps are found between the gonads or between the gonads and the gut loop (Kott, 1969), suggesting that they may be associated with reproduction or digestion. Low biochemical values indicated that the endocarps are unlikely to play a role in nutrient storage.

The high organic content of the body components of C. verrucosa translated into high levels of energy (kJ. g- ~ dry wt), levels similar to those in the tropical colonial ascidian Cystodvtes lobatus (Lambert, 1979) and nine species of Canadian arctic ascidians (Wacasey & Atkinson, 1987). Most of the energy in C. verrucosa was allocated to the tunic and body wall (75Y/o of the total), tissues which may provide protection and nutrient storage. Allocations of energy to the intestines and the mature ovitestes were similar to one another and substantially lower than that allocated to the tunic and body

CHEMICAL DEFENSE OF AN ANTARCTIC ASCIDIAN 171

wall (,~ 10-18% of total energy). The gonad index [(kJ mature ovitestes.total kJ - l ) x 100] of sexually mature individuals was similar to values calculated for plank- totrophic and lecithotrophic antarctic asteroids (McClintock, 1989c). This index should be considered a conservative estimate of reproductive output, as ascidians generally spawn successively over the reproductive period, and individuals may have released some gametes prior to collection.

Several C. verrucosa spawned in the laboratory during this study (November) indicating that at least some of the individuals in the population were sexually mature. Embryos measuring 330/~m in diameter were neutrally or slightly positively buoyant, suggestive of a planktonic lecithotrophic mode of larval development (McClintock, unpubl, data). The energetic content of the body tissues of C. verrucosa was generally 2-3 times that of other sessile antarctic marine invertebrates such as sponges (Dayton et al., 1974) or crinoids (McClintock & Pearse, 1987). These high nutrient and energy levels, combined with the high population density (0.4 ind and 197 kJ .m-2) and the sessile nature of this ascidian, should make C. verrucosa an attractive prey item for antarctic predators such as fish, sea stars, gastropods and nudibranchs.

An exponential equation best describes the relationships between height and body weight in C. verrucosa. Individuals appear to gain height proportionately more rapidly than weight during the early growth phase (up to 6 cm height). This relationship reverses after individuals attain a height of ~ 10 cm, when they begin to gain weight exponen- tially. The relationship between weight and height in other ascidians is poorly known (Millar, 1971). Height and width show a linear relationship in the solitary ascidian Corella willmeriana (Lambert, 1968). Because individuals were not followed over time and lack growth rings, e.g., Chelyosoma macleayanum (Huntsman, 1921), it is difficult to make definitive conclusions about growth rate in C. verrucosa. However, size frequency analysis of the population at Compromise Cove revealed at least four distinct size cohorts. Many antarctic ma~'ia:: " invertebrates have annual reproductive cycles (Pearse et al., 1991), and it is likely that these size cohorts correspond to annual recruitment events. If this is the case, then it would appear that C. verrucosa lives at least 4 yr. Clearly, its life span could be much longer, as the final size cohort may be cumulative, containing individuals which have attained some maximal size.

Life span in most ascidians is comparatively short, lasting from 6 months to 3 yr (Millar, 1971), and has been related to water temperature. For example, Ciona sp. lives only a few months in tropical waters, but may live up to a few years in subarctic regions (Dybern, 1965). However, Styela plicata, a warm water species, lives 5-6 months in warm years and 7-8 months in cold years (Kanatani et al., 1964). Generally, cold water species such as C. vemlcosa live longer than warm water species (Millar, 1971). In Antarctica, where water temperature is a constant - 1.8 °C, a large number of marine invertebrates grow slowly and are long lived (Dayton et al., 1974). Nonetheless, there are species which grow rapidly, e.g., the sponges Mycale acerata and Homaxinella

balfourensis (Dayton et al., 1974; Dayton, 1989), indicating that slow growth is not necessarily a correlate of low water temperature (Pearse et al., 1991). Limited food

172 J.B. McCLINTOCK ET AL.

availability is another factor which has been suggested to cause reduced growth rates in many antarctic marine invertebrates (Clarke, 1991). Svane & Lundalv (1982) argue that a physically stable environment is responsible for the unusually long life=span (> 11 yr) of the cold water ascidian Pyura tessellata.

The tunic of C. verrucosa was rejected by both sympatric and allopatric fish. As allopatric fish models rejected finely ground tunic embedded in agar, it is evident that the tunic is rejected not strictly because of its tough texture, but due to the presence of noxious compound(s). Goodbody (1962) also found that the tunic of Ascidia nigra was distasteful to fish. The tough nature of the tunic could also play a role in providing some protection from potential predators. However, as evidenced by the feeding and egg laying activities of the antarctic opisthobranch gastropod Phyline antarctica, it is possible for predators to penetrate the tunic. Sea stars, common predators of sessile antarctic invertebrates, should be able to extrude their cardiac stomach against the tunic and digest this tissue. While this feeding behavior has never been observed (McClintock et al., pers. obs.; Dayton et al., 1974), sea stars do feed on some temperate and tropical ascidians (Guitierrez & Lay, 1965; Gulliksen & Skjaeveland, 1973; Parry, 1984).

A number of secondary metabolites (Faulkner, 1984; Paul et al., 1990), vanadium (Swinehart etal., 1974; Stoecker, 1980a,b,c), and sulfuric acid (Stoecker, 1978, 1980a,b,c) have all been suggested to play a role in providing ascidians with chemical defense. However, the role of vanadium and sulfuric acid as predator deterrents has been questioned for some ascidians (Parry, 1984). The pH of the homogenized tunic and body wall of C. verrucosa was close to neutral (6.93 and 6.46, respectively) and, therefore, is unlikely to possess specialized bladder cells which secrete sulfuric acid for defense as seen in some ascidians (Stoecker, 1978). Although the tunic was rejected by fish, aqueous tunic extracts were not toxic to sea urchin sperm at the concentrations tested. Moreover, the tunic of C. verrucosa was occasionally lightly fouled with hydroids or bryozoans, indicating that the noxious compound(s) may be sequestered within the tunic or are only moderately effective deterrents against some fouling organisms. Heavily fouled individuals were rarely observed. Kott (1969) also noted that the tunic of C. verrucosa is infrequently fouled with foreign organisms. Future studies need to focus on whether the tunic of C. vern~cosa contains vanadium and/or secondary metabolites which are responsible for its noxiousness.

In addition to the tunic, the endocarps, branchial basket and ovitestes of C. verrucosa

were consistently rejected by the antarctic fish P. borchgrevinki. It is unclear what the significance of distasteful endocarps could be, as the function of these tissues is not known. The noxiousness ofthe branchial basket may be related to its ,oalnerability given its position just below the inhalant siphon (Kott, 1969). However, it should be noted that neither the endocarps nor the branchial basket tissues were consistently rejected by another antarctic fish (7". bernacchii). Rejection of the mature gonads may indicate that planktonic lecithotrophic embryos of C. verrucosa are chemically defended, as seen in the tropical ascidia~ Ecteinascidia turbinata (Young & Bingham, 1987) and in lecithotrophic eggs and embryos of several antarctic sea stars (McClintock & Vernon,

CHEMICAL DEFENSE OF AN ANTARCTIC ASCIDIAN 173

1990). The gonads were mildly acidic (pH - 5.86) and this may have contributed to their rejection. It is unlikely that sperm associated with the ovitestes are chemically defended.

In summary, the solitary ascidian C. verrucosa is abundant in antarctic waters where it is found in clusters of individuals which may attain large size. Recruitment appears to be a reg|dar event and size cohorts suggest a minimum life span of 4 yr. Body components are high in nutrients and energy and given its sessile nature, this species should be a high quality prey item (high nutrient and energy yield per unit foraging effort). Nonetheless, predation levels on C. verrucosa are extremely low. Defense from predation appears to be due, at least in part, to chemical noxiousness of the tunic. Evolutionary selection for chemical defense is not unexpected in a sessile marine invertebrate occurring in an environmentally stable, geologically dated, and biologically accommodated community. The presence ofnoxious chemicals in C. verrucosa adds yet another taxon to a growing list of chemically defended polar marine invertebrates (McClintock et al., 1990).

ACKNOWLEDGEMENTS

We thank S. Duncan for assistance with dissections and J. S. Pearse, R. Rivkin and D. Manahan for use of their study site at Compromise Cove. We also thank the Antarctic Services of ITT, the Antarctic Support Services of the National Science Foundation, and the US Naval Antarctic Support Force for logistic support. This research was supported by NSF Grant No. DPP-8815959 to J.B. McClintock.

REFERENCES

Bakus, G.J., 1974. Toxicity in holothurians: a ~eographic pattern. Biotropica, Vol. 6, pp. 229-236. Bakus, G.J., 1981. Chemical defense mechanisms on the Great Barrier Reef, Australia. Science, Vol. 185,

pp. 497-498. Bakus, G. J. & G. Green, 1974. Toxicity in sponges and holothurians: a geographic pattern. Science, Vol. 185,

pp. 951-953. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of

protein using the principle of protein-dye binding. Anal Biochem., Vol. 72, pp. 248-254. Brody, S., 1945. Bioenergetics and growth. Hafner Publishing, New York, 1023 pp. Ciereszko, L.S., E.M. Ciereszko, E.R. Harris & C.A. Lane, 1963. Vanadium content of some tunicates.

Comp. Biochem. Physiol., Vol. 8, pp. 137-140. Clarke, A., 1991. What is cold adaptation and how should we measure it? Am. Zool., Vol. 31, pp. 81-92. Danskin, G.P., 1978. Accumulation of heavy metals by some solitary tunicates. Can. J. Zool., Vol. 56,

pp. 547-551. Dayton, P.K., 1989. Interdecadal variation in an antarctic sponge and its predators from oceanographic

climate shifts. Science, Vol. 245, pp. 1484-1486. Dayton, P.K., G.A. Robilliard, R.T. Paine & L.B. Dayton, 1974. Biological accomodation in the benthic

community at McMurdo Sound, Antarctica. Ecol. Monogr., Vol. 44, pp. 105-128. Dubois, M., K.A. Gilles, J.K. Hamilton, P.A. Rebers & R. Smith, 1953. Colorimetric determination of

sugars and related substances. Anal, Chem., Vol. 28, pp. 350-356. Dybern, B. I., 1965. The life cycle ofCiona intestinalis (L.) in relation to the environmental temperature. Oikos,

Vol. 16, pp. 109-131.

174 J.B. McCLINTOCK El" AL.

Faulkner, D.J., 1984. Marine natural products: metabolites of marine invertebrates. Nat. Prod. Rep., Vol. 1, pp. 551-598.

Faulkner, D.J., 1986. Marine natural products. Nat. Prod. Rep., Vol. 3, pp. 1-33. Faulkner, D.J., 1987. Marine natural products. Nat. Prod. Rep., Vol. 4, pp. 539-576. Foster, B.A. & J.C. Montgomery, in press. Planktivory in benthic nototheniid fish in MeMurdo Sound,

Antarctica. Environ. Biol. Fish. Freeman, N.K., F.T. Lindgren, Y.C. Ng & A.V. Nichols, 1957. Infrared spectra of some lipoproteins and

related lipids. J. Biol. Chem., Vol. 203, pp. 293-304. Goodbody, I., 1962. The biology ofAscidia nigra (Savigny). I. Survival and mortality in an adult population.

Biol. Bull. (Woods Hole, Mass.), Vol. 124, pp. 40-51. Green, G., 1974. Ecology of toxicity in marine sponges. Mar. Biol., Vol. 40, pp. 207-215. GuUiksen, B. & S.H. Skjaeveland, 1973. The sea starAsterias rubens (L.) as a predator on the ascidian, Ciona

intestinalis (L.) in Borgenfjorden, North-Tr6ndelag, Norway. Sarsia, Vol. 52, pp. 15-20. Gutierrez, M. & S. Lay, 1965. Observaciones Biologicas en la poblacion de Pyura chilensis Molina, 1782,

en Antofagasta (Urochordata: Ascidiacea, Pyuridae). Estud. Oceanol., Vol. 1, pp. 1-31. Huntsman, A.G., 1921. Age-determination, growth and symmetry in the test of the aseidian, Chelyosoma.

Trans. R. Can. Inst., Vol. 13, pp. 27-38. Kanatani, H., T. Hidaka & M. Onosaka, 1964. On the spawning season and growth ofthe ascidian, Styela

plicata in Aburatsubo Bay. Zool. Mag. (Tokyo), Vol. 73, pp. 108-111. Kott, P., 1969. Antarctic ascidiacea. Antarct. Res. Ser., Vol. 13, pp. 1-239. Lambert, G., 1968. The general ecology and growth of a solitary ascidian, Corella wilimeriana. Biol. Bull.

(Woods Hole, Mass.), Vol. 135, pp. 296-307. Lambert, G., 1979. Early post-metamorphic growth, budding and spicule formation in the compound

ascidian Cystodytes lobatus. Biol. Bull. (Woods Hole, Mass.) Vol. 157, pp. 464-477. Lawrence, J.M., 1973. Level, content, and caloric equivalents of the lipid, carbohydrate and protein in the

body components ofLuidia clathrata (Echinodermata : Asteroidea: Platyasterida) in Tampa Bay. J. Exp. Mar. Biol. Ecol., Vol. 11, pp. 263-274.

McCiintock, J.B., 1987. Investigation of the relationship between invertebrate predation and biochemical composition, energy content, spicule armament and toxicity of benthic sponges at McMurdo Sound, Antarctica. Mar. Biol., Vol. 94, pp. 479-487.

McClintock, J. B., 1989a. Toxicity of shallow-water antarctic echinoderms. Polar Biol., Vol. 9, pp. 461-465. McClintock, J.B., 1989b. The biochemical and energetic composition of somatic tissues during growth in

the sea star Pycnopodia helianthoides. Comp. Biochem. Physiol., Vol. 93A, pp. 695-698. McClintock, J.B., 1989c. Energetic composition, reproductive output, and resource allocation in antarctic

asteroids. Polar Biol., Vol. 9, pp. 147-153. McClintock, J. B., J. Heine, M. Slattery & J. Weston, in press. Chemical defense in common shallow-water

antarctic marine invertebrates. Antarct. J.U.S. McClintock, J.B. & J. Jansson, 1990. Pteropod abduction as a chemical defence in a pelagic antarctic

amphipod. Nature (London), Vol. 346, pp. 424-426. McClintock, J. B. & J. S. Pearse, 1987. Reproductive biology of the common antarctic crinoid Promachocrinus

kerguelensis (Echinodermata: Crinoidea). Mar. Biol., Vol. 96, pp. 375-383. McClintock, J.B. & J. Vernon, 1990. Chemical defense in the eggs and embryos of antarctic sea stars

(Echinodermata). Mar. Biol., Vol. 105, pp. 491-495. MiUar, R.H., 1971. The biology of ascidians. Adv. Mar. Biol., Vol. 9, pp. 1-100. Molinski, T.F. & D..I. Faulkner, 1987. Metabolites of the antarctic sponge Dendrilla membranosa. J. Org.

Chem., Vol. 52, pp. 296-298. Moreno, C.A., 1980. Observations on food and reproduction in Trematomus bernacchii (Pisces:Noto-

theniidae) from the Palmer Archipelago, Antarctica. Copeia, Vol. 1, pp. 171-173. Neudecker, S., 1979. Effects of grazing and browsing fishes on the zonation of corals in Guam. Ecology,

Vol. 60, pp. 666-672. Palmer, A.R., 1979. Fish predation and the evolution of gastropod shell sculpture: experimental and

geographical evidence. Evolution, Vol. 33, pp. 697-713. Parry, D. L., 1984. Chemical properties ofthe test of aseidians in relation to predation. Mar, Ecol. Prog. Ser.,

Vol. 17, pp. 279-282. Pearse, J.S., J.B. McClintock & I. Bosch, 1991. Reproduction of antarctic benthic marine invertebrates:

tempos, modes and timing. Am. Zool., Vol. 31, pp. 65-80.

CHEMICAL DEFENSE OF AN ANTARCTIC ASCIDIAN 175

Saito, N. & S.A. Watts, 1989. Activities of hexokinase, phosphofructokinase, and pyruvate kinase in the body wall, pyloric caeca and tube feet of Asterias vulgaris: evidence of body wall as a major source of glycolytic activity. Comp. Biochem. Physiol., Vol. 94B, pp. 263-267.

Smith, M.J. & P.A. Dehnel, 1970. The chemical and enzymatic analyses of the tunic of the ascidian Halocynthia aurantium (Pallas). Comp. Biochem. Physiol., Vol. 35, pp. 17-30.

Stoecker, D., 1978. Resistance ofa tunicate to fouling. Biol. Bull. (Woods Hole, Mass. ), Vol. 155, pp. 615-626. Stoecker, D., 1980a. Distribution of acid and vanadium in Rhopalaea birkelandi Tokioka. J. Exp. Mar. Biol.

Ecol., Vol. 48, pp. 277-281. Stoecker, D., 1980b. Relationships between chemical defense and ecology in benthic ascidians. Mar. Ecol.

Prog. Ser., Vol. 3, pp. 257-263. Stoecker, D., 1980c. Chemical defense of ascidians against predators. Ecology, Vol. 61, pp. 1327-1334. Svane, 1. & T. Lundalv, 1982. Persistence stability in ascidian populations: long-term population dynamics

and reproductive pattern ofPyura tessellata (Forbes) in GuUmarfjorden on the Swedish west coast. Sarsia, Vol. 67, pp. 248-257.

Swinehart, J.H., W.R. Biggs, D.J. Halko & N.C. Schroeder, 1974. The vanadium and selected metal contents of some ascidians. Biol. Bull. (Woods Hole, Mass. ), Vol. 146, pp. 302-312.

Vermeij, G.J., 1978. Biogeography and adaptation. Harvard University Press, Boston, 332 pp. Wacasey, J.W. & E.G. Atkinson, 1987. Energy values of marine benthic invertebrates from the Canadian

Arctic. Mar. Ecol. Prog. Set., Vol. 39, pp. 243-250. Webb, D.A., 1939. Observations on the blood of certain ascidians, with special reference to the biochemistry

of vanadium. J. Exp. Biol., Vol. 16, pp. 499-523. Winston, J. E. & A.W. Bernheimer, 1986. Haemolytic activity in an antarctic bryozoan. J. Nat. Hist., Vol. 20,

pp. 369-374. Young, C.M. & B.L. Bingham, 1987. Chemical defense and aposematic coloration in larvae of the ascidian

Ecteinascidia turbinata. Mar. Biol., Vol. 96, pp. 539-544.