Journal of Chemical Ecology, Vol. 17, No. 7, 1991
A MODIFIED ALLOMONE COLLECTING APPARATUS
B R U C E A. S C H U L T E , I R O C K Y D E N Y S , 2 G E R A L D J. B A K U S , 3'* P H I L I P C R E W S , 4 C L A R K E I D , 4 S T E P H E N N A Y L O R , 4 and L A W R E N C E V. M A N E S 4 ICollege of Environmental Science and Forestry State University of New York Syracuse, New York 13210 2Department of Chemistry and Biochemistry James Cook University of North Queensland Townsville, Q 4812, Australia 3Department of Biological Sciences University of Southern California Los Angeles, California 90089-0371 4Department of Chemistry, Institute of Marine Sciences University of California, Santa Cruz Santa Cruz, California 95064 (Received May 17, t990; accepted March 4, 199l) Abstract--A modified allomone collecting apparatus was designed that could be used under water with a scuba tank. This apparatus provides a simple method of obtaining naturally secreted chemicals from benthic marine organisms at scuba depths, for the first time without the necessity of using a bilge pump. Organic material from Sep-paks in the allomone collector confirmed the release of secondary metabolites from a soft coral into the surrounding water. Key Words--Allomone, Sep-pak, bioactive substances, secondary metabolites, thin-layer chromatography.
INTRODUCTION In the last 20 years it has been r e c o g n i z e d that secondary metabolite synthesis by m a r i n e o r g a n i s m s , p r e v i o u s l y c o n s i d e r e d only a b y - p r o d u c t o f detoxification or an a v e n u e for m e t a b o l i c wastes, is an e v o l u t i o n a r y e v e n t in w h i c h b i o l o g i *To whom correspondence should be addressed. 1327 0098 0331/91/0700-1327$06.50/0 9 1991 Plenum Publishing Corporation
1328
SCHULrEEr AL.
cally active compounds, or bioactive substances, enhance individual survival (Sondheimer and Simeone, 1970; Kittredge et al., 1974; Fenical, 1982). These metabolites are often thought to play an allelopathic role and are known as allelochemicals or allomones. Until the work by Coll et al. (1982), chemical evidence to confirm the presence of marine allomones was lacking. In their study a submersible allomone collecting apparatus, operated by a bilge pump with its battery aboard a boat, was constructed, utilizing Sep-paks (C-18, reverse-phase, silica gel columns) to sample water from around two species of alcyonaceans or soft corals (Sarcophyton crassocaule and Sinulariaflexibilis). Using thin-layer chromatography (TLC), they concluded that the lipophilic secondary metabolites known to exist within these species (i.e., sarcophytoxide and sarcophine from Sarcophyton crassocaule and flexibilide and dihydroflexibilide from Sinularia flexibilis) were also present in the surrounding water. Interestingly, in noncontact transplant experiments involving Sinularia flexibilis with the soft corals Nephthea sp. or Alcyonium molle, no necrotic effects were evidenced. However, over a four-week period all colonies exhibited growth ( " m o v e m e n t " ) away from each other (La Barre et al., 1986). Exudation of bioactive metabolites also was reported for the sponge Aplysina fistularis (Thompson, 1985). The main objective of this study was to develop an allomone collecting apparatus that could be used by scuba divers to collect secondary metabolites secreted from benthic marine organisms. This would allow chemical ecologists a considerable advantage over the system developed by Coll et al. (1982) because the latter were limited to a depth of about 6 m (20 ft) and the apparatus required a battery-powered, submersible bilge pump.
METHODS AND MATERIALS
An allomone collecting apparatus with a hemispherical Plexiglas collecting chamber was designed by one of us (B.A.S.) that could be placed over a sponge or soft coral without additional weights or supports to keep it in position. After extensive laboratory trials at UCSC and field tests over sponges, the apparatus was found to leak. Rather than place large O-rings in the system to prevent the .leakage, a simpler collecting chamber was designed (R.d.N.). Four 18-mmlong hollow stainless-steel tubes 3 mm in diameter with a central opening 1 mm in diameter were brazed to a basal tube 60 mm long. A single 18-mm-long tube was then brazed to the basal tube (Figure 1). This configuration was duplicated and the two pieces (adaptors) connected by four Sep-paks. The lower metal adaptor was connected to an inverted funnel (Figure 2). The upper metal adaptor was connected to an outlet hose that received compressed air from a scuba tank.
1329
ALLOMONE COLLECTING AP.PARATUS
FIG. 1. Close-up photograph of the metal adaptors with Sep-paks in situ.
OUTLET air ~ I N L E T
~..~...-
HOSE HOSE
STEP-UP
(to
buoy)
(from scuba tank)
ADAPTOR
- - S I LICON TUBING
METAL
SEP- PAKS
ADAPTORS
"'~'~--
ST EP-DOW N
ADAPTOR
FUNNEL
FIG. 2. Modified allomone collecting apparatus.
1330
SCHULTE ET AL.
The allomone collecting apparatus is powered by compressed air from a scuba tank, whose flow is controlled by a needle valve in the high-pressure line connected to the low-pressure outlet of a scuba regulator. Water flow rates of 5-8 liters/hr are attainable by adjustment of this needle valve. At a rate of 7 liters/hr and a depth of 8 m (25 ft), the tank loses pressure at an average rate of 150 psi/hr. The allomone device was tested to depths of 13 m (40 ft) without any difficulties. The modified allomone apparatus was first tested in a pool using neutral red. Field tests were carried out at Geoffrey Bay, Magnetic Island, near Townsville, Australia. A small colony of the soft coral Sinulariaflexibilis was enclosed in the collector and cut to ensure release of metabolites. The Sep-paks were primed in MeOH (methanol), and the collector was operated for 4 hr. The Seppaks were removed, returned to the laboratory, and eluted with 20 ml distilled water, 20 ml MeOH, and 20 ml dichloromethane (DCM). The MeOH extract was washed with DCM and the DCM extracts combined. The Sep-paks yielded 25 mg of yellow extract. A sample of the same colony of Sinularia flexibilis was also collected and extracted wet using MeOH and DCM. The MeOH extract was washed with DCM and the DCM extracts combined. A seawater control was run upstream of the soft coral. Comparison of the S. flexibilis extract, the Sep-pak extract, and the pure compounds flexibilide and dihydroflexibilide previously isolated and identified from the soft coral using TLC (Kazlauskas et al., 1978) allowed identification of compounds present in the Sep-pak extract. The TLC was carried out on silica gel (Keisel-gel 60F254) using a 30 % petroleum ether-diethyl ether solvent system. The TLC fractions were visualized using a vanillin-concentrated sulfuric acid spray and the color developed on gentle warming. The Sep-pak extracts of S. flexibilis and the seawater control were examined by [IH]-and [J3C]NMR.
RESULTS
When the modified apparatus was first tested in a pool using neutral red, the Sep-paks removed the neutral red from the water. The organic material eluted from the Sep-paks in the field experiments in Australia was compared with that of the soft coral tissue and the compounds flexibilide and dihydroflexibilide by TLC. Figure 3 confirms, by TLC, the release from S. flexibilis of flexibilide and a number of nonterpenoid compounds thought to be lipids and sterols into the water column. No dihydroflexibilide was detected. [~H] and [13C]NMR further confirmed the presence of lipids and sterols; however, no resonances for either flexibilide or dihydroflexibilide were detected because of low concentrations. TLC and [~H]NMR of the Sep-pak extracts of the upstream seawater control show no flexibilide or dihydroflexibilide to be present. These
1331
ALLOMONE COLLECTING APPARATUS
FIG. 3. Thin-layer chromatographic comparison of the tissue extract (SF), the Sep-pak eluent (SP), and flexibilide (F) of Sinularia flexibiIis; solvent 30% petroleum etherdiethyl ether. results confirm the release of metabolites from S. flexibilis and the ability of the modified allomone apparatus to concentrate organic compounds from the water column.
DISCUSSION
The modified allomone collecting apparatus will allow biologists and chemists to collect natural products (allomones and potential kairomones or pheromones) from marine organisms at any safe diving depth without the need of an accompanying boat containing a battery and cables connected to a submersible pump. The new model (Figures 1 and 2) is simpler and lighter than the original Plexiglas design and does not leak; however, weight does need to be added to the top of the chamber to stabilize it and keep the hood in position over the specimen being tested. The hood can be modified easily to accommodate organisms of different shapes and sizes. This apparatus continued to perform well during extensive field studies on soft corals in Australia conducted by one of us (R.d.N.). Through TLC, the presence of known bioactive second-
1332
SCHULTE ET AL.
ary metabolites was confirmed in the whole-organism extracts of the soft coral species under study and in the water extracts surrounding the animal studied. Examination of organisms other than soft corals would reveal whether allelopathy is a general marine phenomenon or not. Our failure to detect allomones from around marine sponges may be the result of leaks in the Plexiglas allomone model or some other cause (e.g., the sponges sampled may not release allomones). Further studies on sponges with the new model should clarify this issue. The modified allomone collecting apparatus will facilitate the study of waterborne chemical interactions by providing a simple means of sampling the water around aquatic organisms for bioactive substances. Acknowledgments--We thank Drs. John Coil, Porfirio Alifio, Jim Kittredge, and Frederick Browand for making valuable suggestions on the design of the allomone apparatus. Drs. Richard Brusca, Russel Zimmer, and John CoU critically reviewed the manuscript, for which we are grateful. Support for this research was provided by a grant from the University of Southern California Institute for Marine and Coastal Studies (awarded to B.A.S.), by the U.S. Office of Naval Research (grant 00014-84-K-0375 awarded to G.J.B.), and by Sea Grant R/MP-33, U.R.E.P., and Syntex (awarded to P. Crews).
REFERENCES
COLL, J.C., BOWDEN,B.F., TAPIOLAS,D.M., and DUNLAP,W.C. 1982. In situ isolation of allelochemicals released from soft corals (Coelenterata: Octocorallia): A totally submersible sampiing apparatus. J. Exp. Mar. Biol. Ecol. 60:293-299. FENICAL, W. 1982. Natural products chemistry in the marine environment. Science 215:923-928. KAZLAUSKAS,R., MURPHY, P.J., WELLS, R.J., SCHONHOLZER, P., and COLL, J.C. 1978. Cembranoid constituents from an Australian collection of soft coral Sinulariaflexibilis. Aust. J. Chem. 31:1817-1824. KITTREDGE, J.S., TAKAHASHI, F.T., LINDSEY, J., and LASKER, R. 1974. Chemical signals in the sea: Marine allelochemics and evolution. Fish. Bull. 72:1-11. LA BARRE,S.C., COLL, J.C., and SAMMARCO, P.W. 1986. Competitive strategies of soft corals (Coelenterata: Octocorallia). III. Spacing and aggressive interactions between alcyonareans. Mar. Ecol. Prog. Ser. 28:147-156. SONOHEIMER, E., and S1MEONE, J.B. 1970. Chemical Ecology. Academic Press, New York. 336 pp. THOMPSON, J.E. 1985. Exudation of biologically active metabolites in a sponge (Aplysinafistularis) I. Biological evidence. Mar. Biol. 88:23-26.
A MODIFIED ALLOMONE COLLECTING APPARATUS
B R U C E A. S C H U L T E , I R O C K Y D E N Y S , 2 G E R A L D J. B A K U S , 3'* P H I L I P C R E W S , 4 C L A R K E I D , 4 S T E P H E N N A Y L O R , 4 and L A W R E N C E V. M A N E S 4 ICollege of Environmental Science and Forestry State University of New York Syracuse, New York 13210 2Department of Chemistry and Biochemistry James Cook University of North Queensland Townsville, Q 4812, Australia 3Department of Biological Sciences University of Southern California Los Angeles, California 90089-0371 4Department of Chemistry, Institute of Marine Sciences University of California, Santa Cruz Santa Cruz, California 95064 (Received May 17, t990; accepted March 4, 199l) Abstract--A modified allomone collecting apparatus was designed that could be used under water with a scuba tank. This apparatus provides a simple method of obtaining naturally secreted chemicals from benthic marine organisms at scuba depths, for the first time without the necessity of using a bilge pump. Organic material from Sep-paks in the allomone collector confirmed the release of secondary metabolites from a soft coral into the surrounding water. Key Words--Allomone, Sep-pak, bioactive substances, secondary metabolites, thin-layer chromatography.
INTRODUCTION In the last 20 years it has been r e c o g n i z e d that secondary metabolite synthesis by m a r i n e o r g a n i s m s , p r e v i o u s l y c o n s i d e r e d only a b y - p r o d u c t o f detoxification or an a v e n u e for m e t a b o l i c wastes, is an e v o l u t i o n a r y e v e n t in w h i c h b i o l o g i *To whom correspondence should be addressed. 1327 0098 0331/91/0700-1327$06.50/0 9 1991 Plenum Publishing Corporation
1328
SCHULrEEr AL.
cally active compounds, or bioactive substances, enhance individual survival (Sondheimer and Simeone, 1970; Kittredge et al., 1974; Fenical, 1982). These metabolites are often thought to play an allelopathic role and are known as allelochemicals or allomones. Until the work by Coll et al. (1982), chemical evidence to confirm the presence of marine allomones was lacking. In their study a submersible allomone collecting apparatus, operated by a bilge pump with its battery aboard a boat, was constructed, utilizing Sep-paks (C-18, reverse-phase, silica gel columns) to sample water from around two species of alcyonaceans or soft corals (Sarcophyton crassocaule and Sinulariaflexibilis). Using thin-layer chromatography (TLC), they concluded that the lipophilic secondary metabolites known to exist within these species (i.e., sarcophytoxide and sarcophine from Sarcophyton crassocaule and flexibilide and dihydroflexibilide from Sinularia flexibilis) were also present in the surrounding water. Interestingly, in noncontact transplant experiments involving Sinularia flexibilis with the soft corals Nephthea sp. or Alcyonium molle, no necrotic effects were evidenced. However, over a four-week period all colonies exhibited growth ( " m o v e m e n t " ) away from each other (La Barre et al., 1986). Exudation of bioactive metabolites also was reported for the sponge Aplysina fistularis (Thompson, 1985). The main objective of this study was to develop an allomone collecting apparatus that could be used by scuba divers to collect secondary metabolites secreted from benthic marine organisms. This would allow chemical ecologists a considerable advantage over the system developed by Coll et al. (1982) because the latter were limited to a depth of about 6 m (20 ft) and the apparatus required a battery-powered, submersible bilge pump.
METHODS AND MATERIALS
An allomone collecting apparatus with a hemispherical Plexiglas collecting chamber was designed by one of us (B.A.S.) that could be placed over a sponge or soft coral without additional weights or supports to keep it in position. After extensive laboratory trials at UCSC and field tests over sponges, the apparatus was found to leak. Rather than place large O-rings in the system to prevent the .leakage, a simpler collecting chamber was designed (R.d.N.). Four 18-mmlong hollow stainless-steel tubes 3 mm in diameter with a central opening 1 mm in diameter were brazed to a basal tube 60 mm long. A single 18-mm-long tube was then brazed to the basal tube (Figure 1). This configuration was duplicated and the two pieces (adaptors) connected by four Sep-paks. The lower metal adaptor was connected to an inverted funnel (Figure 2). The upper metal adaptor was connected to an outlet hose that received compressed air from a scuba tank.
1329
ALLOMONE COLLECTING AP.PARATUS
FIG. 1. Close-up photograph of the metal adaptors with Sep-paks in situ.
OUTLET air ~ I N L E T
~..~...-
HOSE HOSE
STEP-UP
(to
buoy)
(from scuba tank)
ADAPTOR
- - S I LICON TUBING
METAL
SEP- PAKS
ADAPTORS
"'~'~--
ST EP-DOW N
ADAPTOR
FUNNEL
FIG. 2. Modified allomone collecting apparatus.
1330
SCHULTE ET AL.
The allomone collecting apparatus is powered by compressed air from a scuba tank, whose flow is controlled by a needle valve in the high-pressure line connected to the low-pressure outlet of a scuba regulator. Water flow rates of 5-8 liters/hr are attainable by adjustment of this needle valve. At a rate of 7 liters/hr and a depth of 8 m (25 ft), the tank loses pressure at an average rate of 150 psi/hr. The allomone device was tested to depths of 13 m (40 ft) without any difficulties. The modified allomone apparatus was first tested in a pool using neutral red. Field tests were carried out at Geoffrey Bay, Magnetic Island, near Townsville, Australia. A small colony of the soft coral Sinulariaflexibilis was enclosed in the collector and cut to ensure release of metabolites. The Sep-paks were primed in MeOH (methanol), and the collector was operated for 4 hr. The Seppaks were removed, returned to the laboratory, and eluted with 20 ml distilled water, 20 ml MeOH, and 20 ml dichloromethane (DCM). The MeOH extract was washed with DCM and the DCM extracts combined. The Sep-paks yielded 25 mg of yellow extract. A sample of the same colony of Sinularia flexibilis was also collected and extracted wet using MeOH and DCM. The MeOH extract was washed with DCM and the DCM extracts combined. A seawater control was run upstream of the soft coral. Comparison of the S. flexibilis extract, the Sep-pak extract, and the pure compounds flexibilide and dihydroflexibilide previously isolated and identified from the soft coral using TLC (Kazlauskas et al., 1978) allowed identification of compounds present in the Sep-pak extract. The TLC was carried out on silica gel (Keisel-gel 60F254) using a 30 % petroleum ether-diethyl ether solvent system. The TLC fractions were visualized using a vanillin-concentrated sulfuric acid spray and the color developed on gentle warming. The Sep-pak extracts of S. flexibilis and the seawater control were examined by [IH]-and [J3C]NMR.
RESULTS
When the modified apparatus was first tested in a pool using neutral red, the Sep-paks removed the neutral red from the water. The organic material eluted from the Sep-paks in the field experiments in Australia was compared with that of the soft coral tissue and the compounds flexibilide and dihydroflexibilide by TLC. Figure 3 confirms, by TLC, the release from S. flexibilis of flexibilide and a number of nonterpenoid compounds thought to be lipids and sterols into the water column. No dihydroflexibilide was detected. [~H] and [13C]NMR further confirmed the presence of lipids and sterols; however, no resonances for either flexibilide or dihydroflexibilide were detected because of low concentrations. TLC and [~H]NMR of the Sep-pak extracts of the upstream seawater control show no flexibilide or dihydroflexibilide to be present. These
1331
ALLOMONE COLLECTING APPARATUS
FIG. 3. Thin-layer chromatographic comparison of the tissue extract (SF), the Sep-pak eluent (SP), and flexibilide (F) of Sinularia flexibiIis; solvent 30% petroleum etherdiethyl ether. results confirm the release of metabolites from S. flexibilis and the ability of the modified allomone apparatus to concentrate organic compounds from the water column.
DISCUSSION
The modified allomone collecting apparatus will allow biologists and chemists to collect natural products (allomones and potential kairomones or pheromones) from marine organisms at any safe diving depth without the need of an accompanying boat containing a battery and cables connected to a submersible pump. The new model (Figures 1 and 2) is simpler and lighter than the original Plexiglas design and does not leak; however, weight does need to be added to the top of the chamber to stabilize it and keep the hood in position over the specimen being tested. The hood can be modified easily to accommodate organisms of different shapes and sizes. This apparatus continued to perform well during extensive field studies on soft corals in Australia conducted by one of us (R.d.N.). Through TLC, the presence of known bioactive second-
1332
SCHULTE ET AL.
ary metabolites was confirmed in the whole-organism extracts of the soft coral species under study and in the water extracts surrounding the animal studied. Examination of organisms other than soft corals would reveal whether allelopathy is a general marine phenomenon or not. Our failure to detect allomones from around marine sponges may be the result of leaks in the Plexiglas allomone model or some other cause (e.g., the sponges sampled may not release allomones). Further studies on sponges with the new model should clarify this issue. The modified allomone collecting apparatus will facilitate the study of waterborne chemical interactions by providing a simple means of sampling the water around aquatic organisms for bioactive substances. Acknowledgments--We thank Drs. John Coil, Porfirio Alifio, Jim Kittredge, and Frederick Browand for making valuable suggestions on the design of the allomone apparatus. Drs. Richard Brusca, Russel Zimmer, and John CoU critically reviewed the manuscript, for which we are grateful. Support for this research was provided by a grant from the University of Southern California Institute for Marine and Coastal Studies (awarded to B.A.S.), by the U.S. Office of Naval Research (grant 00014-84-K-0375 awarded to G.J.B.), and by Sea Grant R/MP-33, U.R.E.P., and Syntex (awarded to P. Crews).
REFERENCES
COLL, J.C., BOWDEN,B.F., TAPIOLAS,D.M., and DUNLAP,W.C. 1982. In situ isolation of allelochemicals released from soft corals (Coelenterata: Octocorallia): A totally submersible sampiing apparatus. J. Exp. Mar. Biol. Ecol. 60:293-299. FENICAL, W. 1982. Natural products chemistry in the marine environment. Science 215:923-928. KAZLAUSKAS,R., MURPHY, P.J., WELLS, R.J., SCHONHOLZER, P., and COLL, J.C. 1978. Cembranoid constituents from an Australian collection of soft coral Sinulariaflexibilis. Aust. J. Chem. 31:1817-1824. KITTREDGE, J.S., TAKAHASHI, F.T., LINDSEY, J., and LASKER, R. 1974. Chemical signals in the sea: Marine allelochemics and evolution. Fish. Bull. 72:1-11. LA BARRE,S.C., COLL, J.C., and SAMMARCO, P.W. 1986. Competitive strategies of soft corals (Coelenterata: Octocorallia). III. Spacing and aggressive interactions between alcyonareans. Mar. Ecol. Prog. Ser. 28:147-156. SONOHEIMER, E., and S1MEONE, J.B. 1970. Chemical Ecology. Academic Press, New York. 336 pp. THOMPSON, J.E. 1985. Exudation of biologically active metabolites in a sponge (Aplysinafistularis) I. Biological evidence. Mar. Biol. 88:23-26.
