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Chemical ecology of three antarctic gastropodsJAMES B. MCCLINTOCK, PATRICK J. BRYAN, and MARC SLATTERY, Department of Biology, University ofAlabama,

Birmingham, Alabama 35294-1170BILL J. BAKER, WESLEY Y. YOSHIDA, and MARK HAMANN, Department of Chemistry, Florida Institute of Technology,

Melbourne, Florida 32901JOHN N. HEINE, Moss Landing Marine Laboratories, Moss Landing, California 95039-3304

At depths below the effects of anchor ice and ice scour(Dayton et al. 1969, 1970, pp. 244-258), the antarctic ben-

thos is thought to be structured primarily by biological factorssuch as predation and competition (Dayton et al. 1974). Such"biological accommodation" coupled with an extensive geo-logical history characterized by relatively static environmen-tal conditions (approximately 20 million years; Dayton et al.1994) has provided ample time for the evolution of chemicalmeans of defense in gastropods and other antarctic marineinvertebrates (McClintock el al. 1990, 1991, 1992a, 1994a;Baker et al. 1993). Water-column-dwelling gastropods, suchas pteropods, have likely been subjected to intense predationpressure from pelagic fish. Moreover, although benthic gas-tropods do not appear to be the preferred food of most bot-tom-dwelling fish (Eastman 1993), they are likely prey of theabundant seastars (Dearborn 1977, pp. 293-326; McClintock1994).

As part of our multidisciplinary program investigatingaspects of the chemical ecology of antarctic marine inverte-brates, we have undertaken studies to investigate antarcticgastropods which are likely to possess chemical means ofdefense. Previous studies (McClintock and Janssen 1990;McClintock et al. 1992b) have revealed that whole-body tis-sues of the antarctic pteropod Clione antarctica or mantle tis-sues of the prosobranch mollusk Marseniopsis mollis and thenudibranch Tritoniella belli are rejected by antarctic fish(either Pagothenia borchgi-evinkii or Trematomus bernacchii).Moreover, mantle tissue homogenates of M. mollis and T.belli caused significant sensory tubefoot retractions in fivespecies of antarctic seastars (McClintock et al. 1992b). All ofthese gastropods lack an external shell for defense, although

the prosobranch M. mollis has a vestigial shell imbeddedwithin the mantle tissues. Although these results indicatedthat these antarctic gastropods were noxious to ecologicallyrelevant predators, the secondary metabolites responsible forthis bioactivity were unknown. In this paper, we review thenature of the bioactive compounds and discuss their possibledietary derivation.

The common pteropod C. antarctica was collected usingplankton nets near Hut Point and Cape Armitage in Novem-ber and December 1993. Pteropods were sequentially extract-ed in hexane, chloroform, methanol, and aqueous methanol.The antarctic fish P. borchgrevinki and Pseudo trematom usbernacchii showed feeding deterrence to 2 percent alginatepellets containing 2 percent dried krill and tissue-level con-centrations of the hexane extract [P.1. Bryan, W.Y. Yoshida,J.B. McClintock, and B.J. Baker, unpublished manuscript: Anecological role for pteroenone, a novel antifeedant from theconspicuous antarctic pteropod C. antarctica (Gymnosomata:Gastropoda)]. No feeding deterrence was observed inresponse to pellets containing the other extracts or krill alone.Using flash column chromatography, we further separatedthe hexane fraction, and using high pressure liquid chro-matography, we isolated five pure compounds. These com-pounds were imbedded in alginate pellets and offered to bothantarctic fish. One of the compounds caused rapid rejectionof the krill pellets, and when we used 1- and 2-dimensionalproton and carbon-13 nuclear magnetic resonance (NMR),we determined the compound to be a linear -hydroxyketone(C 14H2402) and named it "pteroenone" (figure 1; Bryan et al.,unpublished manuscript, previously cited; Yoshida et al. inpreparation). The primary prey of C. antarctica, the shelled

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pteropod Limacina helicina (Gilmer and Lalli 1990), did notcontain pteroenone suggesting that this defensive compoundis likely synthesized de novo.

The prosobranch M. mollis and the dorid nudibranch T.belli, as well as their respective primary prey, the tunicateCnemidocarpa verrucosa and the soft coral Clavulariafrankliniana (Dayton et al. 1974), were collected from Octo-ber through December 1993 from approximately 30 meters ofwater near Hut Point, Cape Armitage, or Danger Slopes, nearMcMurdo Station. Multiple individuals (more than 10) of T.belli and C. frankliniana were lyophilized and tissues pooled.The mantle, viscera, and foot of three M. mollis and the tunicof three C. verrucosa were lyophilized and pooled. Dried tis-sues were extracted in organic solvents. An amino acid deriv-ative was obtained by reverse-phase chromatography elutedwith water from the mantle, visceral, and foot tissues ofethanolic extracts of M mollis (McClintock et al. 1994b). A 'HNMR spectral analysis indicated the presence of homarine(figure 2), which was confirmed by high-pressure liquid chro-matography (HPLC) analysis. Shrimp-treated filter paperdisks loaded with tissue-level concentrations of homarine andpresented to the omnivorous antarctic seastar Odontastervalidus caused significant feeding deterrence (McClintock etal. 1994b). No homarine was detected in the tunic of C. verru-cosa. Nonetheless, subsequent HPLC analysis of the epibionts(bryozoans and hydroids) on the surface of the tunic revealedthe presence of homarine suggesting that M. mollis may infact derive its defensive chemistry from these epizooites.

The dorid nudibranch T. belli was extracted in acetoneand the concentrate partitioned between ethyl acetate andwater. Employing normal phase HPLC, the ethyl acetate solu-ble portion yielded three gylcerol ethers, including chimylalcohol (McClintock et al. in press; figure 3), a known antimi-crobial and fish antifeedant (Gustafson and Anderson 1985).Shrimp-treated filter paper disks loaded with one of three logconcentrations of chimyl alcohol (bracketing tissue level con-centration) caused significant feeding deterrence in theomnivorous antarctic seastar 0. validus when compared torejection rates for control shrimp disks (McClintock et al. inpress). An ethyl acetate/hexane fraction of the soft coral T.belli was subjected to normal phase HPLC to yield amongother fatty acid derivatives, chimyl alcohol. The isolatedchimyl alcohol was characterized by comparison with com-mercially available material and by 'H NMR spectroscopy.The verification of chimyl alcohol in the tissues of the softcoral C. frankliniana suggests that the dorid T. belli may bederiving its defensive chemistry from its diet. This needs to beconfirmed by future radioisotope studies.

We thank Mike Davies-Coleman and John D. Faulkner forproviding data on the secondary metabolite chemistry of T.belli. We also wish to thank the Antarctic Support Associates,the Antarctic Support Services of the National Science Foun-dation, and the U.S. Antarctic Support Force for their logisti-cal support. This work was supported by National ScienceFoundation grants OPP 91-18864 and OPP 91-17216 to JamesB. McClintock and Bill J. Baker, respectively.

o OH

PteroenoneFigure 1. Chemical structure of the compound pteroenone, a linearhydroxyketone with fish antifeedant properties isolated from the tis-sues of the antarctic pteropod C/lone antarctica.

"'14^

EM ON-N

ICH3

HomarineFigure 2. Chemical structure of the amino acid derivative homarine, aseastar antifeedant isolated from the foot, mantle, and visceral tissuesof the antarctic prosobranch mollusk Marseniopsis molls.

OH OHOCH3

HOO HOO(CH2)12CH3

Chimyl Alcohol

OH

HOO(CH2)19CH3

Figure 3. Chemical structures of three glycerol ethers isolated fromwhole-body tissues of the antarctic dorid nudibranch Tritoniella be/li.Chimyl alcohol was found to cause feeding inhibition in the omnivo-rous antarctic seastar Odontaster validus.

References

Baker, B.J., W. Kopitzke, M. Hamann, and J.B. McClintock. 1993.Chemical ecology of antarctic sponges from McMurdo Sound,Antarctica: Chemical aspects. Antarctic Journal of the U.S., 28(5),132-133.

Dayton, P.K., B.J. Mordida, and F. Bacon. 1994. Polar marine commu-nities. American Zoologist, 34(1), 90-99.

CO2..

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Dayton, P.K., G.A. Robilliard, and A.L. DeVries. 1969. Anchor ice for-mation in McMurdo Sound, Antarctica, and its biological effects.Science, 245(3864), 1484-1486.

Dayton, P.K., G.A. Robilliard, and R.T. Paine. 1970. Benthic faunalzonation as a result of anchor ice at McMurdo Sound, Antarctica.In M.W. Holgate (Ed.), Antarctic ecology (Vol. 1). London: Academ-ic Press.

Dayton, P.K., G.A. Robilliard, R.T. Paine, and L.B. Dayton. 1974. Bio-logical accommodation in the benthic community at McMurdoSound, Antarctica. Ecological Monographs, 44(1), 105-128.

Dearborn, J.H. 1977. Food and feeding characteristics of antarcticasteroids and ophiuroids. In G. Llano (Ed.), Adaptations withinantarctic ecosystems. Houston: Gulf Publishing.

Eastman, J.T. 1993. Antarctic fish biology. New York: Academic Press.Gilmer, R.W., and G.M. Lalli. 1990. Bipolar variation in Clione, a gym-

nosomatous pteropod. American Malacological Bulletin, 8(1),67-75.

Gustafson, K., and R.J. Anderson. 1985. Chemical studies of BritishColumbia nudibranchs. Tetrahedron, 41(6), 1101-1108.

McClintock, J.B. 1994. The trophic biology of antarctic echinoderms.Marine Ecology Progress Series, 111(1), 191-202.

McClintock, J.B., B.J. Baker, M. Hamann, M. Slattery, R.W. Kopitzke,and J. Heine. 1994a. Tube-foot chemotactic responses of the spon-givorous seastar Perknaster fuscus to organic extracts of antarcticsponges. Journal of Chemical Ecology, 20(4), 859-870.

McClintock, J.B., B.J. Baker, M.T. Hamann, W. Yoshida, M. Slattery,J.N. Heine, P.J. Bryan, G.S. Jayatilake, and B.H. Moon. 1994b.Homarine as a feeding deterrent in the common shallow-water

antarctic lamellarian gastropod Marseniopsis mollis: A rare exam-ple of chemical defense in a marine prosobranch. Journal ofChemical Ecology, 20(10), 2539-2549.

McClintock, J.B., B.J. Baker, M. Slattery, J.N. Heine, P.J. Bryan, W.Toshida, M.T. Davies-Coleman, and D.J. Faulkner. In press. Chem-ical defense of the common shallow-water nudibranch Tritoniellabelli Eliot (Mollusca: Tritoniidae) and its prey, Clavulariafranklini-ana Rouel (Cnidaria: Octocorallia). Journal of Chemical Ecology.

McClintock, J.B., J. Heine, M. Slattery, and J. Weston. 1990. Chemicalbioactivity in shallow-water antarctic marine invertebrates.Antarctic Journal of the U.S., 25(5), 260-262.

McClintock, J.B., and J. Janssen. 1990. Pteropod abduction as a chem-ical defense in a pelagic antarctic amphipod. Nature, 346(6283),462-464.

McClintock, J.B., M. Slattery, J. Heine, and J. Weston. 1991. Density,energy content, and chemical activity of three conspicuousantarctic benthic marine invertebrates. Antarctic Journal of theU.S., 26(5), 172-173.

McClintock, J.B., M. Slattery, J. Heine, and J. Weston. 1992a. Thechemical ecology of the antarctic spongivorous seastar Perknasterfuscus. Antarctic Journal of the U.S., 27(5), 129-130.

McClintock, J.B., M. Slattery, J. Heine, and J. Weston. 1992b. Chemicaldefense, biochemical composition and energy content of threeshallow-water antarctic gastropods. Polar Biology, 11, 623-629.

Yoshida, W., P.J. Bryan, B.J. Baker, and J.B. McClintock. In prepara-tion. Isolation and characterization of pteroenone, the fish feedingdeterrent in the antarctic pteropod Clione antarctica. Journal ofNatural Products.

Chemical constituents of four antarctic sponges inMcMurdo Sound, Antarctica

BILL J. BAKER and WESLEY Y. YOSHIDA, Department of Chemistry, Florida Institute of Technology, Melbourne, Florida 32901JAMES B. MCCLINTOCK, Department of Biology, University ofAlabama at Birmingham, Birmingham, Alabama 35294

Sponges constitute the largest proportion of biomass on thebenthos of McMurdo Sound, Antarctica, occupying 55 per-

cent of free space; all other benthic invertebrates combinedcover only 5 percent of the McMurdo seafloor (Dayton et al.1974). Antarctic sponges are known to be preyed upon byseastars and/or gastropod mollusks, including the seastarsPerknaster fuscus, Odontaster validus, and 0. meridianalisand the mollusks Tritoniella belli, Marseniopsis mollis, andAustrodoris mcmurdensis (Dayton et al. 1974; McClintock etal. 1994a; McClintock in press). In fact, P. fuscus is the majorpredator of sponges, specializing on and regulating the abun-dance of Mycale acerata, though including a small proportionof many other sponges (Dayton et al. 1974).

Based on this central role of Perknasterfuscus in McMur-do Sound sponge ecology, we developed a behavioral bioas-say to evaluate the ability of sponges to deter P. fuscus(McClintock et al. 1990; McClintock et al. 1994b) and foundthat many sponges do in fact yield extracts that exert a behav-ioral effect on P. fuscus, an effect that is likely indicative ofdeterrence (McClintock et al. 1994b). During austral summer1993-1994, we carried out the isolation and characterizationof chemical substances from several McMurdo Sound

sponges and report here on the defensive nature of sub-stances isolated from Isodictya erinacea, Dendrilla membra-nosa, Latrunculia apicalis, and Kirkpatrickia variolosa.

Sponges were collected using scuba between 6 and 40meters depth from Hut Point, Danger Slopes, and Cape Evanson Ross Island, Antarctica. Organisms were freeze-dried, thensubjected to solvent extraction of increasing polarity: hexane,chloroform, methanol, and 70:30 methanol/water (threetimes each). The solvents were removed in vacuo, and theextracts were transferred to tared vials from which aliquotswere removed for bioassay. Active extracts were examined bythin-layer chromatography then subject to repeated high-pressure liquid chromatography (HPLC) separation untilpure. Characterization of purified substances was carried outusing conventional one- and two-dimensional nuclear mag-netic resonance experiments in combination with infraredand mass spectral techniques (Silverstein, Bassler, and Morrill1991). Chemotactic tubefoot response assays were conductedaccording to McClintock et al. (1994b).

The methanol extract of Isodictya erinacea displayedchemotactic tubefoot retraction activity (McClintock et al.1994b). Erebusphenone and p-hydroxybenzaldehyde (figure)

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