Chemical Defense in Ascidians of the Didemnidae ... - ACS Publications

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Bioconjugate Chem. 2003, 14, 30−37

Chemical Defense in Ascidians of the Didemnidae Family† Madeleine M. Joullie´,*,‡ Michael S. Leonard,‡ Padma Portonovo,‡ Bo Liang,‡ Xiaobin Ding,‡ and James J. La Clair§ Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, and Bionic Brothers, Postfach 51 11 07, D-13371 Berlin, Germany. Received July 16, 2002; Revised Manuscript Received October 17, 2002

Fluorescent analogues (DB1 and TA1) of the secondary metabolites didemnin B (DB) and tamandarin A (TA) were synthesized to investigate the potential chemical defense mechanisms of tunicates in the family Didemnidae. These compounds were found to alter predator-prey relations. Five species of freshwater fish and one marine fish, the damselfish Amphiprion ocellaris, were acclimated to a diet of mosquito larvae. Fish showed an immediate, negative reaction to mosquito larvae treated with g5 ng of DB1 or TA1, with consumption of larvae resulting in regurgitation. Both freshwater and marine fish learned to avoid tainted prey by associating species of larvae with “distaste”. Distaste for a given organism also arose when depsipeptides DB1 or TA1 were transferred to the fish from the surrounding medium. Fluorescence microscopy in fish indicated that a similar processing and localization followed ingestion and absorption of DB1 or TA1. Fluorescent labeling of DB or TA provided an ideal tool to conduct short-term studies of predator-prey relationships between fish and marine invertebrate larvae.

INTRODUCTION

Tunicates of the family Didemnidae produce a class of depsipeptides called the didemnins (Rinehart et al., 1981; Sakai et al., 1996; Li and Joullie´, 1992; Rinehart, 2000; Vera and Joullie´, 2002). Didemnin B (DB,1 Figure 1) was one of the first members of this class to show biological activity. Recently, Vervoort et al. (2000) reported the isolation and characterization of tamandarin A (TA, Figure 1) from an unclassified didemnid from a shallowwater reef in Brazil. While the chemical structure and cellular activity of TA is comparable to that of DB, the two metabolites have not yet been shown to exhibit a common biochemical and ecological function. Here we provide further insight into the mechanisms underlying these activities. Modification of a marine natural product (i.e., addition of a fluorescent tag) requires knowledge of its biological and ecological function (Faulkner, 2000a; Faulkner, 2000b; Moore, 1999). Both naturally occurring and synthetic didemnins are known to inhibit protein synthesis, initiate apoptosis, and reduce cell proliferation (Fimiani, 1987; LeGrue et al., 1988; Maldonado et al., * To whom correspondence should be addressed: Madeleine M. Joullie´, Ph.D., University of Pennsylvania, Department of Chemistry, 231 S. 34th St., Philadelphia, PA 19104-6323. Telephone: (215) 898-3158. E-mail: [email protected]. † Dedicated to Professor Koji Nakanishi. ‡ University of Pennsylvania. § Bionic Brothers. 1 Abbreviations: DA ) didemnin A; DACA ) 7-dimethylaminocoumarin-4-acetic acid; DB ) didemnin B; DB1 ) coumarintagged didemnin analogue;  ) extinction coefficient; F ) number of feedings; GI50 ) growth inhibition of 50%; IC50 ) protein synthesis inhibition of 50%; λa ) absorption maximum; λf ) fluorescence maximum; LC50 ) lethal concentration 50%; M ) number of schools; N ) a given population of fish; Φf ) fluorescence quantum yield; PBS ) phosphate buffer solution; S ) school size; SAR ) structure/activity relationships; TA ) tamandarin A; TA1 ) coumarin-tagged tamandarin analogue; TGI ) total growth inhibition.

1982; Montgomery et al., 1987; Rinehart et al., 1982). While the biochemical action of DB has not been fully determined (Beidler et al., 1999; Crews et al., 1994; Crews et al., 1996), a compilation of cellular, affinity and structure-activity relationship studies suggest that protein synthesis inhibition and cell proliferative activities arise through different pathways (Meng et al., 1998). Therefore, it was critical to demonstrate that incorporation of a fluorescent marker preserved the biological activity of the depsipeptides. Using known structure-activity relationships (Rinehart, 2000; Sakai et al., 1996; Vera and Joullie´, 2002), we appended a fluorescently labeled side-chain onto the didemnin and tamandarin macrocycles (Figure 1). A 7-N,N-dimethylaminocoumarin-4-acetic acid (DACA) label was used for this study (Portonovo et al., 2000), as its fluorescence (photophysical properties of probes DB1 and TA1 provided in Table 1) allowed visualization in vivo at concentrations comparable to those naturally expressed by the tunicates (Rinehart et al., 1981; Vervoort et al., 2000). As shown through established assays (Table 1), the didemnin analogue DB1 was better at reducing cell proliferation (GI50 and TGI) than DB, offering activity at less than 1 nM. The activity of TA1 was comparable to that of TA. With these labels in hand, we examined their role in altering predator-prey relations. Lindquist and Hay (1995) found that extracts of Trididemnum solidum (containing DA, DB, as well as other derivatives) deterred predators. In these and preceding studies (Lindquist et al., 1992), DB was shown to be critical in protecting the tunicate larvae (Young and Bingham, 1987; Van Duyl et al., 1981; Ford, 1996). Upon consuming bait containing ∼23 µg of extract mg-1, the pinfish Lagodon rhomboides regurgitated. These fish quickly learned to avoid this bait. Other organisms such as the anemone Aiptasia pallida consumed the depsipeptide-containing food and rapidly lost fitness, as expressed by a profound decrease in growth as well as sexual and asexual reproduction.

10.1021/bc025576n CCC: $25.00 © 2003 American Chemical Society Published on Web 11/27/2002

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Figure 1. Structures of DA, DB, TA, and their fluorescent analogues DB1 and TA1. Structural differences in the macrocycles are accentuated. Table 1. Biological and Photophysical Activity of Selected Depsipeptidesa DB rangeb GI50 5-46 nM TGI 5-270 nM LC50 0.036-50 µM IC50 λa  λf Φf

DBc

TA

13 nM 1.5 nM4 66 nM 3.8 µM 0.89 µM 1.3 µM

DB1c

TA1c

11 nM 295 nM 7.2 µM 16 ( 1.2 µM

49 nM 589 nM 22.4 µM 19 ( 0.9 µM 370 nm 21 000 cm-1 M-1 460 nm 0.19

369 nm 23 200 cm-1 M-1 459 nm 0.21

a GI , TGI, and LC 50 50 were determined by screening through NCI's Developmental Therapeutics Program. Determination of protein synthesis inhibition (IC50) and photophysical properties are described in the methods. b The range provided illustrates the minimum and maximum activity found when screening DB against a series of tumor cell lines. c NCI-60 mean data from NCI60 tumor cell screen. d Activity is measured in pancreatic carcinoma BX-PC3.

These studies showed that the didemnin-containing extracts protect both adult and larval tunicates from fish and invertebrate predators. What remained to be explained was how the tunicates could produce powerful inhibitors of cell proliferation and growth (Table 1) and yet not inhibit their own development. We hypothesized that fish might learn to avoid prey containing didemnins so that, over time, very low tissue concentrations would suffice to deter predation. We synthesized fluorescent analogues of the didemnins and tamandarins so that their uptake by predators of didemnin-containing organisms could be examined using analytically pure samples. To this end, five species of common freshwater fish and

one marine species, the damselfish Amphiprion ocellaris, were chosen for the study of response to the uptake of DB1 and TA1. The reason for using multiple species was to ensure that the findings were generally applicable to fish from a variety of taxa and habitats. We used two species of mosquito larvae as prey and, in a series of experiments, examined the response of predators to both species, when containing the depsipeptides as well as when unprotected by these chemical defense agents. MATERIALS AND METHODS

Fish. Studies were conducted in a laboratory setting from August 1999 to March 2000. A marine system was developed using the damselfish Amphiprion ocellaris. This species was complemented with five freshwater species, all of which lack natural exposure to marine tunicates. Both fresh and marine systems were housed in 100 × 40 × 50 cm tanks (200 L), unless otherwise noted. The systems were recirculated and aerated. Five freshwater species were used: firehead tetra Hemigrammus bleheri (Gery and Mahnert, 1986); peppered corydoras Corydoras paleatus (Jenyns, 1840); common zebrafish Danio rerio (Hamilton, 1822); celebes medaka Oryzias celebensis (Weber, 1894); and glass catfish Kryptopterus bicirrhis (Cuvier and Valenciennes, 1840). H. bleheri and K. bicirrhis were kept at 13 dGH (hardness), pH 6.5 ( 0.3 and 23 ( 3 °C, C. paleatus and D. rerio at 13 dGH, pH 7.0 ( 0.8 and 20 ( 2 °C, O. celebensis at pH 8.5+0.2 and 25 ( 2 °C, and A. ocellaris at 26 ( 2 °C with a salinity of 1.018, pH 8.1 ( 0.1. The media were changed weekly. Freshwater media were maintained at less than 0.05 ppm ammonia, 0.5 ppm of nitrite, and 50.0 ppm of nitrate. The adjustment of the pH was necessary to provide the ideal medium for the given fish. Using the

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intensity of DACA fluorescence (Table 1), the rate of absorption of DB1 and TA1 either through larvae or the medium randomly deviated within 1.2% over the pH range of 5.5-7.5. Under the conditions used in the preceding experimentation, the variance of pH was irrelevant to the transfer of the analogues DB1 and TA1. Studies were conducted on a given population of fish (N) distributed into small schools (M). Each school was held in an individual tank. The school size (S), as given by S ) N M-1, was set to S ) 2 for A. ocellaris, S ) 5 for C. paleatus, S ) 10 for H. bleheri, S ) 10 for K. bicirrhis, S ) 13 for O. celebensis, and S ) 30 for D. rerio, unless otherwise noted. The number of feedings (F) served to calibrate contact between fish (predator) and mosquito larvae (prey). Each fish species was acclimated over 3 weeks (F ) 20) to a diet of mosquito larvae by a daily feeding with Anopheles stephensi and Culex pipiens (referred to herein as acclimation). Each feeding consisted of a 20% excess of larvae in 1:1 ratio of A. stephensi to C. pipiens (i.e., the number of larvae added was 20% more than the number of fish). Feedings lasted for 30 min, at which point the remaining larvae were removed by net. During acclimation, none of the six species of fish favored a given species of mosquito larvae, as determined by calculating the percentage of A. stephensi selected. Over the acclimation period, the percentage of A. stephensi selected was 49.4 ( 6.8 for H. bleheri (N ) 100, M ) 10), 52.7 ( 4.3 for C. paleatus (N ) 50, M ) 10), 50.1 ( 1.2 for D. rerio (N ) 300, M ) 10), 53.2 ( 2.9 for O. celebensis (N ) 26, M ) 2), 49.0 ( 5.6 for K. bicirrhis (N ) 100, M ) 10), and 49.4 ( 6.8 for A. ocellaris (N ) 20, M ) 10). The percentage of the fish that did not consume at each feeding was determined by counting the number of mosquito larvae that remained after feeding. Over repetitive feedings (F ) 50), 96.5% of H. bleheri (N ) 100, M ) 10), 97.5% of C. paleatus (N ) 50, M ) 10), 98.6% of D. rerio (N ) 300, M ) 10), 96.1% of O. celebensis (N ) 26, M ) 2), and 97.8% of K. bicirrhis (N ) 100, M ) 10), and 95.9% of A. ocellaris (N ) 20, M ) 10) consumed at least a single larva per feeding. Mosquito Larvae. A. stephensi and C. pipiens larvae were selected with a length of 10-12 mm and weight of 8-12 mg. This size is ∼6-12 times that of the larval tunicate Trididemnum solidum (Lindquist and Hay, 1995; Lindquist et al., 1992). While DB is not uniformly distributed throughout the adult tunicate (Rinehart, 2000), one can estimate the average tissue concentration based on the amount of metabolite isolated per mass of tunicate. For DB and TA, this concentration lies between 50 and 100 ppm. Since the larval mimics employed in this study were larger than tunicate larva, we charged each mosquito larva with 50 ( 4 ng of DB1 or TA1, providing an average tissue concentration of ∼4-6 ppm. This was accomplished by gently shaking each larva for 5 min in sterile Eppendorf tubes containing 50 µL of 42 nM DB1 (or 45 nM TA1) diluted in 1 mL of sterilized mineral water. This transfer was verified by measuring the amount of analogue absorbed from the medium (using fluorescence spectroscopy) and/or by tissue analysis (method described below). By linearly adjusting the solution concentration of DB1 or TA1, this procedure could be used to load larvae with 10 ( 2 to 250 ( 12 ng larva-1. Molecular Tracking System. The transmission between prey and predator was monitored using fluorescence from DB1 and TA1. Physiological processing of these ligands was examined on an Axiovert 100 with a conventional Zeiss DAPI filter set with excitation, G365; beam splitter, FT 395; emission, LP 420. Images were

Joullie´ et al.

Figure 2. K. bicirrhis. Physiological processing after ingestion of a C. pipiens larva containing 50 ( 4 ng of DB1. Images provided in two views: white light (left) and fluorescence (right). Abbreviations: liver (L), kidney (K), pancreas (P), stomach (S), intestine (I), epurals (EP), hypurals (H1-5), hermal spines of the preural centra (HPU2), epurial joint (JE), neural spines of the preural centra (NPU3), preural centra (PU1), parhypural (PH), ural centrum (U1), and uroneural (UN), neural spine (NS), hemal spine (HS), spinal chord (SC), vertebral centrum (VC), notochord (N). (a) Dorsal view of the digestive system. (b) Lateral view of stomach and intestine. In addition to the digestive system, DB1 also localizes within the skeleton. (c) Lateral view of the caudal fin. (d) Close-up of an epural joint. (e) Lateral view of posterior vertebrate. Comparable images were also produced by ingestion of a larva containing 50 ( 4 ng of TA1 or exposure for 5 min to 100 mL of media containing 10 ( 2 ng of either DB1 or TA1.

collected on a Zappa digital camera. Physiological examinations were performed after dissection of a random subsample of 3-5 fish per school. Advantageously, the lack of pigment in Kryptopterus bicirrhis allowed direct investigation in vivo. Images from this fish are provided in Figure 2. The localization studies were controlled by

Chemical Defense in Ascidians

comparing transfer with that of N-methyl-7-N,N-dimethylaminocoumarin-4-acetamide (i.e., a nondepsipeptide containing derivative of DACA). This derivative, unlike DB1 and TA1, provided weak nonspecific staining in fish tissue, therein further verifying that localization of fluorescence shown in Figure 2 arose from the depsipeptide-function of DB1 and TA1. Furthermore, a “no treatment” negative control revealed that any intrinsic fluorescence was below the limit of detection. Tissue Concentration Analysis. Tissue concentrations of DB1 or TA1 were determined by ultramicrosonicating ∼10 mg of tissue (either mosquito larvae or fish) in 30 µL of phosphate buffer solution (PBS). The concentration of depsipeptide in the resulting sample was determined fluorometrically using the DACA label (excitation at λmax ) 370 nm and emission at λmax ) 460 nm, see Table 1). Rejection Studies. After acclimation, colonies of fish were given mosquito larvae containing DB1 or TA1 (exposed larvae). The fish were allowed to feed on the larvae for 30 min, at which point excess larvae were collected using a net, and counted. After removal, the fish were again fed with an excess of a 1:1 mixture of nontreated C. pipiens and A. stephensi larvae. This method was used to collect data presented in Figures 3-8. Larval Success Study. Modified 10 L tanks were constructed with a 2 mm mesh barrier that divided the vessel in two equal volumes. This barrier restricted fish to one side of the tank, while allowing larvae to freely access the entire tank. Larvae were added in a single transfer to the side of the tank containing the fish. Twenty larvae (either C. pipiens or A. stephensi larvae) were challenged to avoid consumption by fish (S ) 20). Larvae that crossed to the right side of the tank were removed and tallied (referred to as escaped). Without prior exposure to DB1 or TA1, naive D. rerio (N ) 400, M ) 20, S ) 20) hunted 98 ( 0.5% of the larvae before they crossed the barrier (control). These fish were then removed and individually exposed for 30 min to 100 mL of media containing 50 ( 4 ng of DB1 followed by a 5 min wash in 100 mL of depsipeptide-free media. The ability of these fish to learn was determined by counting the number of larvae that escaped and comparing with control. The data from this study is depicted in Figure 9. Fish Diversity Study. Thirty tanks (200 L) were loaded with Danio rerio (N ) 600, M ) 30) and Kryptopterus bicirrhis (N ) 600, M ) 30). Each tank contained 20 D. rerio and 20 K. bicirrhis. The D. rerio were acclimated and pretrained over 10 feedings to avoid C. pipiens larvae, while the K. bicirrhis were only acclimated. Each of the tanks was fed over a period of 30 d (F ) 30) with a diet consisting of 30 C. pipiens containing 50 ( 4 ng of DB1 and 20 depsipeptide-free A. stephensi. Five minutes after each feeding, excess mosquito larvae were removed and the stomachs of the fish examined. Fish that failed to hunt a depsipeptide-free A. stephensi (i.e., consume no larvae or a fluorescent C. pipiens) were marked with a drop of paint. Fish that failed three times were removed from the tank. The results of this study are presented in Figure 11. RESULTS

Biochemical Analyses. Cell proliferation and protein synthesis assays were conducted on DB1 and TA1 to ensure proper biological activity. The effects of DB1 and TA1 on cell growth and proliferation were determined

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via the NCI-60 tumor cell screen (Boyd and Paul, 1995). Protein synthesis inhibition was measured in MCF-7 cells using a protocol described by Toogood (Ahuja et al., 2000; Beidler, 1999). The results of these assays are summarized in Table 1. Transfer by Consumption. Given its favorable lack of pigmentation, physiological studies began with Kryptopterus bicirrhis. Individual K. bicirrhis were fed with a larva containing 50 ( 4 ng of DB1 or TA1 (exposed larva) and monitored over the course of 10 d. Immediately after swallowing the exposed larva, fluorescence from DB1 or TA1 crossed the stomach wall of the fish. Soon thereafter, fluorescence spread throughout the stomach and entered the intestine. This path was followed by regurgitation. Within 15 min of this regurgitation, fluorescence from DB1 was no longer apparent in the stomach, but remained within the intestine (Figure 2a,b). At a dose of 50 ( 4 ng larva-1, fluorescence from DB1 remained in the intestine for up to 3 d. The rate of transfer, physiological processing, and intensity of localization could not be differentiated between DB1 and TA1 on Kryptopterus bicirrhis. The amount of DB1 or TA1 required to cause regurgitation was then determined by measuring the residual concentration of depsipeptide in regurgitated mosquito larvae (using tissue concentration analysis, see methods). After 65 regurgitations in D. rerio, 42 ( 3 ng of DB1 remained within the mosquito larva, indicating that each D. rerio regurgitated after absorbing a dose less than 8 ( 2 ng of DB1. Under identical conditions, 12 ( 4 ng of TA1 was transferred. The increased dose of TA1 may be explained by its reduced biological activity (Table 1). Comparable analysis in the other fish indicated that H. bleheri absorbed 8 ( 2 ng DB1 and 11 ( 3 ng TA1; C. paleatus, 8 ( 2 ng DB1 and 12 ( 2 ng TA1; O. celebensis, 7 ( 3 ng DB1 and 9 ( 3 ng TA1; K. bicirrhis, 5 ( 3 ng DB1 and 8 ( 2 ng TA1; and A. ocellaris, 10 ( 2 ng DB1 and 19 ( 4 ng TA1. In addition to localization in the digestive system, fluorescence from DB1 and TA1 was also observed in the skeleton of all six fish species. This staining became visible 2 min after consumption and lasted for over a week. As illustrated Figure 2c-e, DB1 showed particular affinity for the endoskeleton at the joints of the eplurals and hypurals. In the spine (Figure 2e), there was a clear affinity for the notochord. The depsipeptides did not bind neural tissue, as even faint DACA fluorescence was not seen in the spinal chord, nerves, or motor neurons. Staining was seen not only in the caudal fin and spine but also in the jaw, chest, pelvic girdle, and dorsal fin. We then compared this delivery with transfer of the depsipeptide from the medium. The rate and physiological accumulation of depsipeptides DB1 or TA1 in K. bicirrhis upon exposure for 5 min to 100 mL of media containing 10 ( 2 ng of DB1 or TA1 was indistinguishable from that observed after ingestion of an exposed larvae containing 50 ( 4 ng of DB1 or TA1 (Figure 2). When transmitted from the medium, fluorescence from DB1 was initially observed in the skin, gills, and mouth of K. bicirrhis. Within minutes, this fluorescence disappeared and again localized in the intestine and joints. This observation suggests that the physiological target of DB1 and TA1 is independent of the digestive system. It also further indicates that the staining in Figure 2 arises from intact DB1 or TA1, and not from products of digestion or metabolism. Calibrating Rejection. A series of experiments were conducted to examine the response of fish to the analogues. As shown in Figure 3, rejection was readily

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Figure 3. Rejection of DB1. Fish given a C. pipiens larvae containing 50 ( 4 ng of DB1 quickly learn to avoid the bait. The data are presented as the percentage of the school that did not consume a larva at each feeding. Six species are shown: H. bleheri (9, N ) 100, M ) 10), C. paleatus ([, N ) 50, M ) 5), D. rerio (b, N ) 300, M ) 10), O. celebensis (0, N )26, M ) 2), K. bicirrhis (], N ) 100, M ) 10), and A. ocellaris (O, N ) 20, M ) 10).

Figure 4. Rejection of TA1. Fish given C. pipiens larvae containing 50 ( 4 ng of TA1 learn to avoid the bait. The data are presented as the percentage of the school that did not consume a larva at each feeding. Six species are shown: H. bleheri (9, N ) 100, M ) 10), C. paleatus ([, N ) 50, M ) 5), D. rerio (b, N ) 300, M ) 10), O. celebensis (0, N )26, M ) 2), K. bicirrhis (], N ) 100, M ) 10), and A. ocellaris (O, N ) 20, M ) 10).

reproduced when mosquito larvae were dosed with 50 ( 4 ng of DB1 or TA1. Over 50% of all six species of fish avoided C. pipiens larvae containing 50 ( 4 ng of DB1 after 5 feedings (Figure 3). After 10 feedings, only ∼5% of these fish continued to consume the exposed larvae. A comparable outcome was observed with TA1 (Figure 4). The intensity of this rejection increased with dose of depsipeptide (Figure 5). When loaded at 10 ( 2 ng larva-1, 20 ( 1% of the Danio rerio (N ) 300, M ) 10) avoided the exposed larvae after two feedings. When enhanced to 50 ( 4 ng larva-1, the rejection after two feedings improved only slightly to 25 ( 2%. A comparable response was seen in the other species of fish. Over the same regime (10 ( 2 ng larva-1 vs 50 ( 4 ng larva-1), the number of H. bleheri (N ) 100, M ) 10) deterred after 2 feedings improved from 17 ( 2% to 23 ( 3%, in C. paleatus (N ) 40, M ) 4) from 18 ( 3% to 26 ( 2%, in O. celebensis (N ) 26, M ) 2) from 19 ( 5% to 21 ( 4%, in K. bicirrhis (N ) 100, M ) 10, N ) 10) from 16 ( 3% to 28 ( 3%, and in A. ocellaris (N ) 20, M ) 13) from 17 ( 6% to 24 ( 4%. While dosages above 10 ( 2 ng larva-1 modestly improved the induction of rejection, fluorescent examinations (Figure 2) indicated that the bulk of this effect arose from increasing the rate of analogue transfer. Fluorescent studies indicated that increasing the dose of DB1 above 50 ( 4 ng larva-1 did not alter the intensity of fluores-

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Figure 5. Danio rerio. Concentration profile. Increasing concentration of DB1 in the larvae increases the rejection of C. pipiens. These data was collected by repeating Figure 3 with 0 ng (9), 10 ( 2 ng (2), 50 ( 4 ng (b), and 250 ( 12 ng (XXXXX) of DB1 per larva.

cence observed in the joints of fish, rather, it enhanced the rate at which DB1 was absorbed in (or transferred to) the stomach of the fish. Given a larva that is 10 mg, 50 ( 4 ng larva-1 corresponds to 50 ppm, a concentration that lies at the lower end of that known to be expressed in tunicate tissue (i.e. 50-100 ppm). While this transfer could operate for the protection of adults, a single didemnid larvae is 0.8-1 mg (Lindquist and Hay, 1995). At this size a single 3 g fish would have to swallow at least 10 larvae to feel the effects of these agents. A curious facet of these tunicate metabolites lies in their expression in the larval stage (Lindquist et al., 1992; Lindquist and Hay, 1995). In the preceding study, we found that the optimal tissue expression of DB1 and TA1 was around 10 ( 2 ng larvae-1. At this expression, critical cell proliferation in the tunicate’s larvae would also be at risk (i.e., anti-proliferative activity occurs at the pM range, Table 1), raising the question of how the tunicate and its larvae express the required amounts of depsipeptide without inhibiting their own growth. Consequently, we examined how predators (fish) assimilated the biochemical information provided by the chemical defense agents. Learned Avoidance. Lindquist and Hay (1995) verified that fish learned to avoid the “distaste” of DB (Hay, 1996). While learning is well documented in fish, we were interested in understanding how this response was processed. We found that the toxicity of DB1 and TA1 was associated with the identity of the prey and not by a chemosensing (Rittschof, 1993). Danio rerio trained to avoid C. pipiens larvae avoided the larvae even when they did not contain DB1 (Figure 6). Not surprisingly, D. rerio trained to avoid C. pipiens readily consumed larvae from a second species, A. stephensi (Figure 7). Danio rerio trained to avoid C. pipiens however had to relearn to avoid A. stephensi containing either DB1 or TA1 (Figure 8). These observations indicate that D. rerio learned to avoid the depsipeptide by processing the structural or behavioral trait(s) of the larvae. This advantageously allowed fish to minimize their contact with the analogues. Indirect Transmission. While not yet examined in the field, logistic analysis suggests that release mechanisms are also feasible. When a 3.8 g Amphiprion ocellaris was exposed to 200 ng of DB1 in 10 L of media for 5 min, the level of fluorescence from DB1 in its joints was indistinguishable from that shown in Figure 2. At this level of transfer, tunicates would have to deliver 20 ng L-1 to their local medium. In a sea with a depth of 10 m and a density of 50 adult tunicates m-2, a single tunicate would need to release 4 µg of depsipeptide

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Figure 6. Danio rerio. Rejection and learning. Schools of zebrafish (N ) 300, M ) 10) learned to reject C. pipiens containing 50 ( 4 ng of DB1 over 5 daily feedings and continued to reject C. pipiens not containing DB1 thereafter. Data presented by plotting the percentage of zebrafish that did not consume a larva over a total of 10 feedings.

Figure 7. Danio rerio. Rejection and acceptance. Schools of zebrafish (N ) 300, M ) 10) that learned to reject C. pipiens containing 50 ( 4 ng of DB1 over 5 daily feedings accepted DB1free A. stephensi (feedings 6-10). Data presented depicts the percentage of zebrafish that did not consume a larva over a total of 10 feedings.

Figure 9. Danio rerio. Larval success study. (top) Zebrafish (N ) 300, M ) 10) in media without DB1 feed normally. (middle) Zebrafish (N ) 300, M ) 10) that were individually exposed to 100 mL media containing 50 ( 4 ng of DB1 after the fifth feeding lost their ability to hunt depsipeptide-free C. pipiens. This loss was not permanent, as shown by the reduction in escaped larvae. (bottom) A comparable outcome was also observed by exposure to 50 ( 4 ng of TA1 after the fifth feeding. Figure 8. Danio rerio. Rejection and relearning. Schools of zebrafish (N ) 300, M ) 10) that learned to reject C. pipiens containing 50 ( 4 ng of DB1 over 5 daily feedings had to relearn to avoid A. stephensi containing 50 ( 4 ng of DB1 (feedings 6-10). Data presented depicts the percentage of zebrafish that did not consume a larva over a total of 10 feedings.

(typically 50-250 µg of depsipeptide are isolated per tunicate). Here the colonial nature of these tunicates could be used to further concentrate the release and targeting of the metabolites. We also examined the possibility of media transfer by examining the appetite of the fish after exposures to media-born DB1. As shown in Figure 9, fish quickly lost their appetite after exposure to 0.5 ng mL-1 of DB1. Dose relation studies indicated that this effect required an exposure of 5 min in media containing 80 pM, or 0.1 ng mL-1 of DB1. While temporary, this loss of appetite allowed the mosquito larvae to escape consumption (i.e., crossing the barrier). Interestingly, fish exposed to media

containing DB1 apparently improved their recognition of DB1-exposed C. pipiens (Figure 10). Danio rerio that only had brief contact to media containing DB1 learned to avoid the exposed C. pipiens much faster than fish that had no prior contact to DB1. We then examined how media transfer could alter the competition between two species of fish. A total of five tanks were prepared with an equivalent number of D. rerio (N ) 100, M ) 5) and K. bicirrhis (N ) 100, M ) 5). Each tank was fed daily with 30 C. pipiens containing 50 ( 4 ng of DB1 and 20 unexposed A. stephensi larvae. When examined without training, D. rerio and K. bicirrhis competed for bait at the same rate and success (not shown). The schools of D. rerio were then removed and trained over 10 feedings to avoid C. pipiens. As indicated in Figure 11, D. rerio excelled at catching the depsipeptide-free A. stephensi, while K. bicirrhis continued to consume the exposed C. pipiens. We then modeled the effects that this competition would have on short-term decline, by tagging each fish

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number of successful or remaining K. bicirrhis was four less than that of the trained D. rerio. After 16 feedings (average presented graphically), no K. bicirrhis remained. DISCUSSION

Figure 10. Danio rerio. Fish learn from media exposure. Exposure to media containing DB1 improved D. rerio’s ability to avoid exposed C. pipiens. Schools of D. rerio trained by exposure to media containing 50 ( 4 ng of DB1 ([, N ) 300, M ) 10) were more apt to avoid treated C. pipiens than naı¨ve D. rerio (9, N ) 300, M ) 10). This experiment was conducted using the conditions described in the larval success study (see methods). The percent of C. pipiens larvae that escaped is shown.

Figure 11. D. rerio and K. bicirrhis. Fish diversity study. The influence of DB1 on an artificial ecosystem containing schools of D. rerio (N ) 200, M ) 10) and K. bicirrhis (N ) 200, M ) 10) was calibrated by examining the ability to hunt. Feedings were conducted with C. pipiens containing 50 ( 4 ng of DB1 and depsipeptide-free A. stephensi. Fish that failed to hunt A. stephensi were marked with paint. Once they failed three times (three dots of paint), they were removed from the tank. (top) D. rerio, trained to avoid DB1, out lasted (bottom) naive K. bicirrhis. The members of each species remaining are displayed as those who did not consume a larva (black), caught a C. pipiens (gray), and caught an A. stephensi (stripes).

There was a rapid, definitive response by six species of fish to larval mosquitoes containing analogues DB1 or TA1. Transfer of the chemical-defense agent from prey (mosquito larvae) to predator (fish) was rapid. Once localized in the intestine and skeleton, DB1 and TA1 were not cleared for several days. In all six species of fish, the processing of DB1 and TA1 was indistinguishable with respect to the kinetics of transfer and thermodynamic localization, as all species of fish displayed a comparable rate of absorption as well as physiological processing. The intensity of this response was sufficient to cause 50% of the fish that consumed larvae containing 50 ( 4 ng of depsipeptide to avoid the treated larvae after only five daily feedings. Subsequent analyses indicated that this response arose after transfer of only 5-10 ng of depsipeptide. The effect was dose dependent and led to association of the larval species with distaste. Thus, once conditioned, the fish did not consume untreated larvae of the same species but fed upon another species of mosquito larvae. The analog-mediated effect did not require ingestion. Transfer of the analogue from the medium to the fish also resulted in a decreased efficiency of hunting. Furthermore, the physiological localization of the analogues in fish was not altered by this mode of delivery. Predator fitness potentially increases with their ability to recognize treated larvae. Fish that had been preconditioned to avoid the depsipeptide-treated larvae were able to hunt other species of larvae selectively and benefit; whereas, unconditioned fish had to learn to avoid the tainted larvae before they could effectively compete for food. This study is among the first to show that TA has similar activity to DB (Liang et al., 2001). Analogues DB1 and TA1 shared a common potency and localization in the predators. The two species of family Didemnidae that produce DB and TA, while from remote geographic locations, use a common method to defend themselves and their otherwise vulnerable larvae by producing two structurally similar but not identical depsipeptides. The results of this study might be used to investigate predator-prey interactions in marine species other than tunicates. ACKNOWLEDGMENT

We thank W. Mu¨ller, W. Rettig, and B. Mallioux for their assistance. This work has been supported in part by grants from the National Institutes of Health (CA40081) and the National Science Foundation (CHE 9901449). This work was conducted under protocols that met standards of the National Institutes of Health, and the countries where experimentation was conducted. LITERATURE CITED

after it failed to hunt properly. Tanks were prepared with 20 D. rerio trained to avoid C. pipiens and 20 untrained K. bicirrhis. Each of the tanks was provided over a period of 30 d (F ) 30) with a diet consisting of 30 C. pipiens containing 50 ( 4 ng of DB1 and 20 depsipeptide-free A. stephensi. After 20 feedings, an average of 11 ( 3 trained D. rerio (N ) 100, M ) 20) remained in each school. The naı¨ve K. bicirrhis (N ) 100, M ) 20) were not as successful (Figure 11). Even after five feedings, the

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