BILL J. BAKER and THOMAS L. BARLOW, Department of Chemistry, Florida Institute of Technology, Melbourne, Florida 32901
JAMES B. MCCLINTOCK, Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-1170
Biological interactions of invertebrates on the benthos of McMurdo Sound, Antarctica, have received considerable attention over the last three decades. Beginning with seminal studies of Dayton, Robilliard, and Paine (1970) and Dayton et al. (1974) characterizing the community structure, subsequent studies have focused on chemical ecological interactions (reviewed by McClintock and Baker 1997). The field of chemical ecology seeks to understand biological interactions that are chemically mediated.
The chemical basis for such ecological interactions lies in secondary metabolic pathways. Metabolites derived from the secondary metabolic pathways differ from primary metabolites in that they are not involved in life-sustaining processes such as energy transport and storage, respiration, and reproduction. Such compounds generally are not ubiquitous. Secondary metabolites, also known as natural products, were once regarded as superfluous, evolutionary dead ends but are now widely regarded as playing a role in ecological interactions of the producing organism (Williams et al. 1989; Eisner and Meinwald 1995).
Our interest in the secondary metabolites present in McMurdo Sound organisms has been driven by an interest in their functional role. Although we have investigated a number of marine phyla, sponges have proven to be a rich source of natural products suitable for an evaluation of their functional role. During our 1997 field season, we had the opportunity to collect specimens of the sponge Suberites sp., and we report here our preliminary chemical ecological evaluation of this common benthic invertebrate.
Lipophilic and hydrophilic extracts of several pooled Suberites sp. were prepared by exhaustive extraction of the freeze-dried specimens with methanol/dichloromethane (1:1) and methanol/water (1:1). The extracts were examined by proton (1H) nuclear magnetic resonance (NMR) spectroscopy, which revealed the presence of a relatively high concentration of terpenoid components in the lipophilic extract. The lipophilic extract was then fractionated by normal phase gradient flash chromatography, producing a fraction eluting in 8:2 hexane/ethyl acetate that contained the terpenoid NMR signals. Further purification of the terpenes was achieved by reversed phase high-performance liquid chromatography (HPLC), yielding two compounds ( 1 and 2 , figure) in a ratio of 3 to 1.
The major component ( 1 ) displayed 1H NMR signals characteristic of several functional groups, including two olefinic protons, two protons on carbons bearing oxygen, an acetoxyl methyl, a vinyl methyl, and four aliphatic methyl groups. The carbon-13 (13C) NMR spectrum contained 27 carbon signals including four olefinic, one ester/amide/acid, and one ketone/aldehyde carbon. The DEPT (distortionless enhancement by polarization transfer) spectrum indicated two olefinic carbons were protonated and two quaternary; the remaining carbons included six methyl, seven methylene, five methine, and five quaternary signals.
Structure elucidation of this isolate was achieved by analysis of two-dimensional NMR spectra including correlation spectroscopy (COSY) and heteronuclear multiple quantum coherence spectroscopy (HMQC). Partial structures representing ring D and parts of rings A and C were generated from analysis of the COSY spectrum. These partial structures were sufficiently unique to narrow the focus of our analysis to two compounds previously reported from an antarctic Suberites species collected from King George Island, suberitenone A (Shin et al. 1995). Comparison of the NMR spectral data of suberitenone A with our isolate 1 established their identity. Similar comparison of our isolate 2 confirmed its identity with suberitenone B.
We investigated the functional role of the suberitenones in two ecological bioassays. The tube-foot retraction assay (McClintock et al. 1994) is designed to detect the ability of a sponge to deter predation by production of toxic or noxious secondary metabolites. The spongivorous sea star Perknaster fuscus , the major sponge predator in McMurdo Sound (Dayton et al. 1974), was used as the test animal in the tube-foot retraction bioassay. In the disk diffusion assay, which provides data to evaluate the ability of sponge chemistry to deter microbial fouling or infection, antibiotic tester strains (ATSs) were employed as a general indicator of microbial cytotoxicity and microbes isolated from McMurdo Sound benthic invertebrates and substratum as indicators of ecologically relevant antibiotic activity. The eight ATS strains included Gram-positive Bacillus subtillus, B. cerius, Staphylococcus aureus , and Micrococcus luteus ; Gram-negative Pseudomonas aeriginosa and Escherichia coli ; yeast Saccharomyces cerevisiae ; and fungi Aspergillus niger . The invertebrate-associated microorganisms, which are currently undergoing identification, include one isolate each from the homogenized tissues of the hydroid Halecium arboreum ; the sponges Latrunculia apicalis , Calyx arcuarius , and Dendrilla membranosa ; and the sea star Acodontaster conspicuus , as well as three microbes from substratum rocks. The original report of the suberitenones failed to find bioactivity in these unusual sesterterpenes.
Results of the bioassays suggest the suberitenones are mediators of chemical defense in Suberites sp. in McMurdo Sound. Suberitenone A ( 1 ) caused a mean 33.6-second [standard deviation (SD)=14.8, number (n)=10] sustained tube-foot retraction and suberitenone B (2), a 29.4-second retraction (SD=18.2, n=10). Control experiments (n=20 each) were consistently shorter in duration: mechanical, 3.6-second retraction (SD=9.0); and silicone matrix, 4.1-second retraction (SD=11.3). A feeding stimulant control used in our prior study (McClintock et al. 1994) was shown to elicit a response indistinguishable from the mechanical and silicone matrix controls; thus, such tube-foot retraction behavior as seen with suberitenones A and B is indicative of feeding deterrence (McClintock et al. 1994).
Suberitenone A ( 1 ) displayed modest activity against two of the ecologically relevant microorganisms. Isolate 28.2, from Halecium arboreum , showed a 3-millimeter (mm) zone of inhibition at 500 micrograms per disk (g/disk) and isolate 22.3, from Acodontaster conspicuus , a 1-mm zone at 1,000 g/disk. Suberitenone B (2) was less active against the same two isolates, displaying a 1-mm zone of inhibition against both at 1,000 g/disk. No antibiotic activity was found in ATS strains. Because the suberitenones have activity against sympatric bacteria, they have the potential to mediate fouling and infection in Suberites sp.
We are grateful to J. Mastro, C. Moeller, and C. Amsler for assistance with collection of sponges; to C. Amsler for isolation of antarctic microbes; and to Robert van Soest for sponge identification. This work was supported by National Science Foundation grants OPP 95-26610 and OPP 95-30735 to Bill J. Baker and James B. McClintock, respectively, and by logistics provided by the Antarctic Support Associates and the U.S. Naval Support Force Antarctica.
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