Chemical ecology of antarctic sponges from McMurdo Sound, Antarctica
Chemical ecology of antarctic sponges from McMurdo Sound, Antarctica: Ecological aspects and MARC SLATFERY, Department of Biology, University ofAlabama, Birmingham, Alabama BILL J. BAKER, Department of Chemistry, Florida Institute of Technology, Melbourne, Florida 32901 JOHN N. HEINE, Moss Landing Marine Laboratory, Moss Landing, California 95039
JAMES B. McCUNT0cK
he incidence of chemical bioactivity in marine inverteT brates has received increasing attention in temperate and tropical fauna (reviewed by Bakus, Targett, and Schulte 1986). Early studies examining the toxicity of marine invertebrates (primarily sponges and holothurians) concluded that the incidence of defensive chemicals was higher at tropical than temperate latitudes (Bakus and Green 1974; Green 1977; Bakus and Thun 1979; Bakus 1981). These studies indicated that sessile and sluggish marine invertebrates commonly contain defensive chemicals, as has been well established in marine and especially terrestrial plants (Harbourne 1977; Rosenthal and Janzen 1979; Steinberg and Paul 1990; Van Alstyne and Paul 1990). Tropical-temperate comparisons led to a latitudinal hypothesis, suggesting an inverse correlation between the incidence of chemical defense in marine invertebrates and latitude (Bakus and Green 1974). This "latitudinal gradient" in chemical defense is thought to be driven by predation pressure, with tropical sessile and sluggish marine invertebrates under proportionately higher levels of predation by browsing fish (Bakus and Green 1974; Vermeij 1978). Therefore, in an evolutionary context, selective pressure for defensive compounds would be expected to be highest at low latitudes and lowest at high latitudes. Nonetheless, recent cytotoxicity (Blunt et al. 1990) and behavioral (McClintock et al. 1991) bioassays conducted with antarctic sponges and other sessile or sluggish antarctic marine invertebrates have detected an incidence of biochemical activity commensurate with temperate marine environments. We have undertaken to isolate and identify bioactive substances from antarctic marine invertebrates and report here our work on antarctic sponges during the 1992 austral summer. In our investigation of chemical ecology of antarctic sponges, we have evaluated extracts from sponges for the presence of defensive substances using a standard antimi crobial assay as well as an ecological assay. We prepared extracts of the freeze-dried sponges with solvents of increasing polarity (sequentially, hexane, chloroform, methanol, and aqueous methanol), thus achieving separation of potential interfering agents (that is, substances that are not secondary metabolites but that might be bioactive, such as salts, large peptides, amino acids, and/or nucleotides) into the latter fraction. The microorganisms used in our antimicrobial assay included a gram-positive bacterium (Bacillus subtilis), a gram-negative bacterium (Escherichia col), two yeasts (Saccharomyces cerevisiae and Aspergillus niger). Petri dishes containing antibiotic medium 1 (bacteria) or dextrose agar (yeast) were inoculated from slants (Presque Isle Cultures). We
applied 5 milligrams (mg) of each extract to 6-millimeter (mm) paper filter disks and ordered these seven or eight per petridish. Zones of inhibition (in mm) were recorded after 24 hours of incubation at 37°C. Chloramphenicol and penicillin G were used as positive controls. Our ecological assay consisted of turning over a sea star in a crystallization dish filled with sea water. The initial response of a sea star when it is turned over is to extend its tubefeet in an effort to locate a solid substrate to grab hold of to assist in righting itself. These tubefeet are small suction apparatuses on the bottom of each arm and are chemosensory, especially those at the tips of the arms. This chemosensi tivity is fundamentally a defensive response (Sloan 1980). Sea star tubefeet are primary sites for perception of chemical stimuli (Lawrence 1975; Sloan and Campbell 1981; McClintock, Klinger, and Lawrence 1984), so they are very sensitive "instruments" for the detection of defensive substances. We took advantage of this sensitivity by applying our extracts to a glass rod and positioning this rod near the retracted tubefeet such that when a tubefoot emerged, it would encounter our extract-coated rod (McClintock et al. in press). Deterrent substances cause the tubefoot to retract, as do feeding stimulants; however, after the initial retraction in response to a feeding stimulant, the tubefoot is almost immediately reextended, whereas in the case of a deterrent substance, no retraction persists (we measured retractions up to the 60 seconds we allotted for each trial). We controlled for mechanical stimulation (a clean rod) and the effects of the silicon grease matrix in which the extract was imbedded (rod with silicon grease). Additionally, we employed a feeding stimulant for each experiment (fish extract in silicon grease coated on a glass rod). Each extract, control, and stimulant was examined 10 times, and each was blind to the observer. We chose the antarctic sea star Perknasterfuscus because it is a general spongivore and the chief predator on antarctic sponges. The antimicrobial assay (table) showed that most of the activity was in the chloroform extract (56 percent of extracts tested), which is the most likely fraction to contain functionalized, small molecular weight compounds. Of the hexane extracts, which were composed largely of sterols and fatty acids, only 20 percent showed activity, whereas 30 percent of the methanol extracts were active. In the tubefoot retraction assay, hexane sponge extracts also elicited the lowest levels of significant tubefoot responses, with only 39 percent of the sponge species tested showing activity. In contrast, chloroform and methanol extracts elicited a significant tubefoot retraction response in 73 percent and 78 percent of the species tested, respectively. These two
ANTARCTIC JOURNAL - REVIEW 1993 134
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Bioactivity
of antarctic
References sponge extracts(Z
TFR TFR TFR LIy. rcum TFR TFR, BS Cinachyra antarctica TFR TFR Dendrilla membranosa - TFR TFR Gellius benedeni - TFR BS - BS Gellius tenella* TFR TFR Haliclona sp. BS Haliclona dancoi TFR,BS Homaxonella balfourensis - TEA TER TFA Inflate/la be/li TEA, BS lsodictya erinacea BS Isodictya setifera' - BS
TFR TFR TER TER BS
BS Kirkpatrickia variolosa - TEA - , TER,BS,EC BS Latrunculia apicalis TEA,BS,EC BS Leucetta leptorhapsis EC Mycale acerata - - TFA BS Polymastia invaginata TFR,BS - TFR Rose/la nuda BS Rose/la racovitzae TEA Sphaerotylus antarcticus - TER,BS - ", BS,SC Tetilla leptoderma
TEA TEA BS TEA
aTEA: Significant tubefoot retraction, BS:
Bacillus subtilis, EC: Escherichia coIl, SC: Saccharomyces cerevisiae, * denotes no
data on tubefoot retraction are available; - denotes no bioactivity in assays conducted.
assays together suggest bioactive metabolites from antarctic sponges are of a polar nature. Although it remains to be determined that secondary metabolites are responsible for all of the activity we are reporting, bioactive secondary metabolites have already been isolated from a number of these antarctic sponges (Baker et al., Antarctic Journal, in this issue). It maybe of some ecological significance that the only two rapidly growing sponges, Homaxinella balfourensis and Mycale acerata, either were not bioactive or had very low bioactivity and that one of these sponges, M. acerata, is the primary dietary item of Perknaster
fuscus.
We would like to thank J. Maestro for assistance with the collections of the sponges. The Antarctic Support Associates, Inc., the Antarctic Support Services of the National Science Foundation, and the U.S. Naval Antarctic Support Force provided logistical support. This research was supported by National Science Foundation grant OPP 91-18864 to J.B. McClintock and OPP 91-17216 to B.J. Baker.
Baker, B.J., R. 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). Bakus, G.J. 1981. Chemical defense mechanisms on the Great Barrier Reef, Australia. Science, 211(4481), 497-498. Bakus, G.J., and G. Green. 1974. Toxicity in sponges and holothurians: A geographic pattern. Science, 185(4155), 951-953. Bakus, G.J., N.M. Targett, and B. Schulte. 1986. Chemical ecology of marine organisms: An overview. Journal of Chemical Ecology, 12, 951-987. Bakus, G.J., and M.A. Thun. 1979. Bioassays on the toxicity of Caribbean sponges. In C. Levi and N. Bourny-Esnault (Eds.), Sponge biology (Vol. 291). Paris: Centre National de la Recherché Scientifique (C.N.R.S.). Blunt, J.W., M.H.G. Munro, C.N. Battershill, B.R. Copp, J.D. McCombs, N.B. Perry, M. Prinsep, and A.M. Thompson. 1990. From the antarctic to the antipodes: 45° of marine chemistry. New Journal of Chemistry, 14, 751-775. Green, G. 1977. Ecology of toxicity in marine sponges. Marine Biology, 40(3), 207-215. Harbourne, J.D. 1977. Introduction to ecological biochemistry. New York: Academic Press. Lawrence, J.M. 1975. On the relationship between marine plants and sea urchins (Echinodermata: Echinoidea). Oceanographic Marine BiologyAnnual Review, 13, 213-286. McClintock, J.B., B.J. Baker, M. Slattery, M. Hamann, R. Kopitzke, and J. Heine. In press. Chemotactic tubefoot responses of the spongivorous sea star Perknaster fuscus to organic extracts from antarctic sponges. Journal of Chemical Ecology. McClintock, J.B., J. Heine, M. Slattery, and J. Weston. 1991. Chemical defense in shallow-water antarctic marine invertebrates. Antarctic Journal of the U.S., 25(5), 260-262. McClintock, J.B., T.S. Klinger, and J.M. Lawrence. 1984. Chemoreception in Ludida clathrata (Echinodermata: Asteroidea): Qualitative and quantitative aspects of chemotactic responses to low molecular weight compounds. Marine Biology, 84(1), 47-52. Rosenthal, G.A., and D.H. Janzen. 1979. Herbivores: Their interaction with secondary plant metabolites. New York: Academic Press. Sloan, N.A. 1980. Aspects of the feeding biology of asteroids. Oceanographic Marine BiologyAnnual Reviews, 18,57-124. Sloan, N.A., and A.C. Campbell. 1981. Perception of food. In M. Jangoux and J.M. Lawrence (Eds.), Echinoderm nutrition. Rotterdam: Balkema Press. Steinberg, P.D., and V. Paul. 1990. Fish feeding and chemical defense of tropical brown algae in Western Australia. Marine Ecology Progress Series, 58(3), 253-259. Van Alstyne, K.L., and V.1. Paul. 1990. The biogeography of polyphenolic compounds in marine macroalgae: Temperate brown algal defenses deter feeding by tropical herbivorous fishes. Oecologia, 84(2),158-163. Vermeij, G.J. 1978. Biogeography and adaptation. Boston: Harvard University Press.
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JAMES B. McCUNT0cK
he incidence of chemical bioactivity in marine inverteT brates has received increasing attention in temperate and tropical fauna (reviewed by Bakus, Targett, and Schulte 1986). Early studies examining the toxicity of marine invertebrates (primarily sponges and holothurians) concluded that the incidence of defensive chemicals was higher at tropical than temperate latitudes (Bakus and Green 1974; Green 1977; Bakus and Thun 1979; Bakus 1981). These studies indicated that sessile and sluggish marine invertebrates commonly contain defensive chemicals, as has been well established in marine and especially terrestrial plants (Harbourne 1977; Rosenthal and Janzen 1979; Steinberg and Paul 1990; Van Alstyne and Paul 1990). Tropical-temperate comparisons led to a latitudinal hypothesis, suggesting an inverse correlation between the incidence of chemical defense in marine invertebrates and latitude (Bakus and Green 1974). This "latitudinal gradient" in chemical defense is thought to be driven by predation pressure, with tropical sessile and sluggish marine invertebrates under proportionately higher levels of predation by browsing fish (Bakus and Green 1974; Vermeij 1978). Therefore, in an evolutionary context, selective pressure for defensive compounds would be expected to be highest at low latitudes and lowest at high latitudes. Nonetheless, recent cytotoxicity (Blunt et al. 1990) and behavioral (McClintock et al. 1991) bioassays conducted with antarctic sponges and other sessile or sluggish antarctic marine invertebrates have detected an incidence of biochemical activity commensurate with temperate marine environments. We have undertaken to isolate and identify bioactive substances from antarctic marine invertebrates and report here our work on antarctic sponges during the 1992 austral summer. In our investigation of chemical ecology of antarctic sponges, we have evaluated extracts from sponges for the presence of defensive substances using a standard antimi crobial assay as well as an ecological assay. We prepared extracts of the freeze-dried sponges with solvents of increasing polarity (sequentially, hexane, chloroform, methanol, and aqueous methanol), thus achieving separation of potential interfering agents (that is, substances that are not secondary metabolites but that might be bioactive, such as salts, large peptides, amino acids, and/or nucleotides) into the latter fraction. The microorganisms used in our antimicrobial assay included a gram-positive bacterium (Bacillus subtilis), a gram-negative bacterium (Escherichia col), two yeasts (Saccharomyces cerevisiae and Aspergillus niger). Petri dishes containing antibiotic medium 1 (bacteria) or dextrose agar (yeast) were inoculated from slants (Presque Isle Cultures). We
applied 5 milligrams (mg) of each extract to 6-millimeter (mm) paper filter disks and ordered these seven or eight per petridish. Zones of inhibition (in mm) were recorded after 24 hours of incubation at 37°C. Chloramphenicol and penicillin G were used as positive controls. Our ecological assay consisted of turning over a sea star in a crystallization dish filled with sea water. The initial response of a sea star when it is turned over is to extend its tubefeet in an effort to locate a solid substrate to grab hold of to assist in righting itself. These tubefeet are small suction apparatuses on the bottom of each arm and are chemosensory, especially those at the tips of the arms. This chemosensi tivity is fundamentally a defensive response (Sloan 1980). Sea star tubefeet are primary sites for perception of chemical stimuli (Lawrence 1975; Sloan and Campbell 1981; McClintock, Klinger, and Lawrence 1984), so they are very sensitive "instruments" for the detection of defensive substances. We took advantage of this sensitivity by applying our extracts to a glass rod and positioning this rod near the retracted tubefeet such that when a tubefoot emerged, it would encounter our extract-coated rod (McClintock et al. in press). Deterrent substances cause the tubefoot to retract, as do feeding stimulants; however, after the initial retraction in response to a feeding stimulant, the tubefoot is almost immediately reextended, whereas in the case of a deterrent substance, no retraction persists (we measured retractions up to the 60 seconds we allotted for each trial). We controlled for mechanical stimulation (a clean rod) and the effects of the silicon grease matrix in which the extract was imbedded (rod with silicon grease). Additionally, we employed a feeding stimulant for each experiment (fish extract in silicon grease coated on a glass rod). Each extract, control, and stimulant was examined 10 times, and each was blind to the observer. We chose the antarctic sea star Perknasterfuscus because it is a general spongivore and the chief predator on antarctic sponges. The antimicrobial assay (table) showed that most of the activity was in the chloroform extract (56 percent of extracts tested), which is the most likely fraction to contain functionalized, small molecular weight compounds. Of the hexane extracts, which were composed largely of sterols and fatty acids, only 20 percent showed activity, whereas 30 percent of the methanol extracts were active. In the tubefoot retraction assay, hexane sponge extracts also elicited the lowest levels of significant tubefoot responses, with only 39 percent of the sponge species tested showing activity. In contrast, chloroform and methanol extracts elicited a significant tubefoot retraction response in 73 percent and 78 percent of the species tested, respectively. These two
ANTARCTIC JOURNAL - REVIEW 1993 134
35294
Bioactivity
of antarctic
References sponge extracts(Z
TFR TFR TFR LIy. rcum TFR TFR, BS Cinachyra antarctica TFR TFR Dendrilla membranosa - TFR TFR Gellius benedeni - TFR BS - BS Gellius tenella* TFR TFR Haliclona sp. BS Haliclona dancoi TFR,BS Homaxonella balfourensis - TEA TER TFA Inflate/la be/li TEA, BS lsodictya erinacea BS Isodictya setifera' - BS
TFR TFR TER TER BS
BS Kirkpatrickia variolosa - TEA - , TER,BS,EC BS Latrunculia apicalis TEA,BS,EC BS Leucetta leptorhapsis EC Mycale acerata - - TFA BS Polymastia invaginata TFR,BS - TFR Rose/la nuda BS Rose/la racovitzae TEA Sphaerotylus antarcticus - TER,BS - ", BS,SC Tetilla leptoderma
TEA TEA BS TEA
aTEA: Significant tubefoot retraction, BS:
Bacillus subtilis, EC: Escherichia coIl, SC: Saccharomyces cerevisiae, * denotes no
data on tubefoot retraction are available; - denotes no bioactivity in assays conducted.
assays together suggest bioactive metabolites from antarctic sponges are of a polar nature. Although it remains to be determined that secondary metabolites are responsible for all of the activity we are reporting, bioactive secondary metabolites have already been isolated from a number of these antarctic sponges (Baker et al., Antarctic Journal, in this issue). It maybe of some ecological significance that the only two rapidly growing sponges, Homaxinella balfourensis and Mycale acerata, either were not bioactive or had very low bioactivity and that one of these sponges, M. acerata, is the primary dietary item of Perknaster
fuscus.
We would like to thank J. Maestro for assistance with the collections of the sponges. The Antarctic Support Associates, Inc., the Antarctic Support Services of the National Science Foundation, and the U.S. Naval Antarctic Support Force provided logistical support. This research was supported by National Science Foundation grant OPP 91-18864 to J.B. McClintock and OPP 91-17216 to B.J. Baker.
Baker, B.J., R. 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). Bakus, G.J. 1981. Chemical defense mechanisms on the Great Barrier Reef, Australia. Science, 211(4481), 497-498. Bakus, G.J., and G. Green. 1974. Toxicity in sponges and holothurians: A geographic pattern. Science, 185(4155), 951-953. Bakus, G.J., N.M. Targett, and B. Schulte. 1986. Chemical ecology of marine organisms: An overview. Journal of Chemical Ecology, 12, 951-987. Bakus, G.J., and M.A. Thun. 1979. Bioassays on the toxicity of Caribbean sponges. In C. Levi and N. Bourny-Esnault (Eds.), Sponge biology (Vol. 291). Paris: Centre National de la Recherché Scientifique (C.N.R.S.). Blunt, J.W., M.H.G. Munro, C.N. Battershill, B.R. Copp, J.D. McCombs, N.B. Perry, M. Prinsep, and A.M. Thompson. 1990. From the antarctic to the antipodes: 45° of marine chemistry. New Journal of Chemistry, 14, 751-775. Green, G. 1977. Ecology of toxicity in marine sponges. Marine Biology, 40(3), 207-215. Harbourne, J.D. 1977. Introduction to ecological biochemistry. New York: Academic Press. Lawrence, J.M. 1975. On the relationship between marine plants and sea urchins (Echinodermata: Echinoidea). Oceanographic Marine BiologyAnnual Review, 13, 213-286. McClintock, J.B., B.J. Baker, M. Slattery, M. Hamann, R. Kopitzke, and J. Heine. In press. Chemotactic tubefoot responses of the spongivorous sea star Perknaster fuscus to organic extracts from antarctic sponges. Journal of Chemical Ecology. McClintock, J.B., J. Heine, M. Slattery, and J. Weston. 1991. Chemical defense in shallow-water antarctic marine invertebrates. Antarctic Journal of the U.S., 25(5), 260-262. McClintock, J.B., T.S. Klinger, and J.M. Lawrence. 1984. Chemoreception in Ludida clathrata (Echinodermata: Asteroidea): Qualitative and quantitative aspects of chemotactic responses to low molecular weight compounds. Marine Biology, 84(1), 47-52. Rosenthal, G.A., and D.H. Janzen. 1979. Herbivores: Their interaction with secondary plant metabolites. New York: Academic Press. Sloan, N.A. 1980. Aspects of the feeding biology of asteroids. Oceanographic Marine BiologyAnnual Reviews, 18,57-124. Sloan, N.A., and A.C. Campbell. 1981. Perception of food. In M. Jangoux and J.M. Lawrence (Eds.), Echinoderm nutrition. Rotterdam: Balkema Press. Steinberg, P.D., and V. Paul. 1990. Fish feeding and chemical defense of tropical brown algae in Western Australia. Marine Ecology Progress Series, 58(3), 253-259. Van Alstyne, K.L., and V.1. Paul. 1990. The biogeography of polyphenolic compounds in marine macroalgae: Temperate brown algal defenses deter feeding by tropical herbivorous fishes. Oecologia, 84(2),158-163. Vermeij, G.J. 1978. Biogeography and adaptation. Boston: Harvard University Press.
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