E .G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160
143
ability of scleractinian corals to inhibit the settlement of invertebrate larvae is only starting to be investigated.
The soft corals Sinularia flexibilis and Sarcophyton glaucum secrete toxic secondary metabolites into the water Coll et al., 1982. Settlement of scleractinian larvae around
these two species were inhibited, suggesting an allelopathic function for the secreted compounds Maida et al., 1995a,b. Bioactive compounds have been detected in
scleractinians Fusetani et al., 1986; Rashid et al., 1995; Koh, 1997, some of which could function as allelochemicals. In a study of the antimicrobial activity of extracts
from 100 scleractinian species, Tubastraea faulkneri inhibited the most species of microbes Koh, 1997. Tubastraea faulkneri may also produce compounds that are toxic
to coral competitors. T
. faulkneri is a non-zooxanthellate coral of the family De- ndrophylliidae and it inhabits overhangs and vertical surfaces. It has a reported depth
range of 3–7 m Wells, 1982, but it occurs from shallow reef flats down to at least 15 m personal observations. This slow-growing species shares the shallower parts of its
range with other faster growing zooxanthellate species of corals, and therefore is likely to experience interspecific competition. The use of allelochemicals that inhibit settlement
and growth of coral competitors would aid T
. faulkneri in persisting among the faster growing species.
The present study investigated the hypothesis that a coral can produce chemicals that are toxic to the larvae of other coral species that are potential competitors. We examined
the effects of natural products from Tubastraea faulkneri on its own larvae and the larvae of 11 other sympatric coral species. The specific aims of this study were: a to
determine if natural metabolites produced by T . faulkneri are deleterious to larvae of
other scleractinian coral species, b to evaluate the likelihood of larvae encountering these compounds at relevant concentrations in their natural environment, and c to
characterize the bioactive compounds in T . faulkneri.
2. Materials and methods
2.1. Extract preparation Tubastraea faulkneri
colonies were collected from Davies Reef 188 509 S, 1478 409 E, Great Barrier Reef, Australia. Specimens were frozen immediately for
storage and transportation. In a pilot study, crude extracts of T . faulkneri were prepared
from five samples taken from separate coral colonies. Absolute ethanol was used for extraction because it is a good solvent and is miscible with water that was present in the
frozen specimens. The extracts were tested on the planulae of Acropora formosa Dana and Platygyra sinensis Edwards and Haime. This showed that natural products of T
. faulkneri were toxic to these two species of coral larvae, so more specimens of T
. faulkneri were collected to prepare a batch extract for testing on coral larvae from a
wider range of species. To prepare the batch extract, fresh specimens of Tubastraea faulkneri were freeze-
dried and weighed before being extracted sequentially in three solvents: dichlorome- thane, methanol and distilled water. This procedure divided the metabolites of T
.
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.G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160
faulkneri into three coarse fractions according to their polarities, from the non-polar dichloromethane extract to the highly polar distilled water extract. Each extract was
dried under vacuum and weighed. To determine the weight of coral tissue from which the metabolites were extracted, the remaining coral was bleached to remove the tissue,
rinsed carefully with distilled water and freeze-dried completely before weighing again to obtain the skeleton weight. The weight of coral tissue calculated by subtracting
skeleton weight from weight of whole coral was then used to determine natural extract concentrations within the coral tissue.
Antimicrobial activity was used as a representative measure of bioactivity in the dichloromethane, methanol and distilled water extracts of Tubastraea faulkneri. The
three extracts and subsequent fractions were screened for activity against a broad range of microbes used in a previous large-scale survey of scleractinian bioactivity Koh,
1997. These microbes were Vibrio alginolyticus Miyamoto et al., V . harveyi Johnson
and Shunk, V . parahaemolyticus Fujino et al., Photobacterium damsela Love et al.,
Alteromonas rubra Gauthier, Synechococcus sp. and Staphylococcus aureus Rosenbach. The first six species were chosen because they were isolated from Australian waters and
are therefore likely to be encountered by T . faulkneri. S. aureus is terrestrial and served
as an indicator for broader spectrum bioactivity. A . rubra was isolated from a coral
Streiner, 1990; P . damsela and V. harveyi are pathogenic to some marine organisms
Fouz et al., 1992; Sutton and Garrick, 1993. V . alginolyticus and V. parahaemolyticus
occur in bacterial films on marine substrates Kaneko and Colwell, 1975; Belas and Colwell, 1982. Synechococcus sp. is a cyanobacterium common in seawater and marine
sediments Glover, 1985. The tests indicated that activity was present almost exclusive- ly in the methanol extract Table 1. Because toxicity to coral larvae can only be tested
during a brief period each year when planulae are available, priority was given to testing the methanol extract.
2.2. Rearing competent coral planulae Eleven species of scleractinians were selected, representing a range of habitat
preferences and competitive abilities. The species belong to four families: Acroporidae: Montipora digitata Dana, Acropora formosa, A
. millepora Ehrenberg, A. pulchra
Table 1
a
Antimicrobial bioassay of crude extracts from Tubastraea faulkneri tested at 500 mg per disc Bioassay organism
Inhibition zone of extracts mm Dichloromethane
Methanol Distilled water
Vibrio alginolyticus 3.0
Vibrio harveyi 2.8
Vibrio parahaemolyticus Photobacterium damsela
3.8 Alteromonas rubra
5.0 Staphylococcus aureus
2.8 Synechococcus sp.
1.5 13.0
a
N 5 5 for each treatment combination. Solvent controls had no activity.
E .G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160
145
Brook, A . tenuis Dana, Faviidae: Favia pallida Dana, Goniastrea aspera Verrill,
Platygyra daedalea, P . sinensis, Fungiidae: Fungia fungites Linnaeus and Pectiniidae:
Oxypora lacera. Only competent larvae were used because they are more likely to encounter active concentrations of allelochemicals from established colonies during the
process of settling on a reef. Pre-competent larvae or embryos are assumed to inhabit the water column away from adult corals.
The larvae were obtained from corals at Magnetic Island 198 89 S, 1468 499 E and Orpheus Island 188 329 S, 1468 289 E during three mass spawnings using methods
developed by Babcock and Heyward 1986. Gravid colonies of broadcast spawning species were collected during the day and kept in large containers of seawater. When
gametes were released at night, they were collected and mixed with those from other colonies of the same species. The resulting larvae were incubated at their natal reefs in
floating plastic containers fitted with plankton mesh lids to ensure close approximation to natural growth conditions. Larvae of most broadcast spawning corals of the Great
Barrier Reef reach competence when they are 4–7 days old Babcock and Heyward, 1986. The larvae used in the toxicity assays were 4–5 days old.
To determine the effects of Tubastraea faulkneri extracts on conspecific larvae, adult colonies were collected 1 week after the main spawning event and maintained in
flow-through aquaria. The brooded larvae were released about 2 weeks after the main spawning event and were competent within a day, settling readily in the aquaria.
Day-old larvae were used for the assays.
2.3. Toxicity bioassay The toxicity of Tubastraea faulkneri extract to scleractinian planulae was determined
for a range of concentrations that were 100 or more times below the concentration in T .
faulkneri tissues. The highest test concentration was made up by dissolving a known weight of the T
. faulkneri extract in a small volume of 80 ethanol and adding the appropriate volume of sea water. Ethanol formed only 1.6 of this solution. Lower
extract concentrations were obtained by serial dilution with seawater, minimizing the error that could arise when pipetting small aliquots of a volatile liquid.
In the pilot experiment, ethanolic extracts from five separate Tubastraea faulkneri colonies were tested on Platygyra sinensis and Acropora formosa planulae at five
21
extract concentrations 31.25, 62.5, 125, 250, and 500 mg ml seawater. Five replicate
glass scintillation vials Wheaton Scientific, NJ were prepared for each concentration, each containing 10 ml of solution. A parallel set of solutions containing seawater with
the same amounts of ethanol as in each extract concentration served as solvent controls. Ten planulae of one species were introduced to each vial using a Pasteur pipette. This
ratio of ten planulae to 10 ml of solution was previously used by Aceret et al. 1995. Treatment and control vials received the planulae in random order. The proportion of
dead planulae in each vial after 4 h was taken as a measure of toxicity of the extract or ethanol at a particular concentration. In order to verify that the small volume of water
used in each vial was sufficient for ten planulae, one species P
. sinensis was retained in the solvent control vials for a week. These larvae experienced an average mortality of
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.G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160
Fig. 1. Toxic effects of Tubastraea faulkneri extracts from five separate colonies on the planulae of Platygyra sinensis and Acropora formosa. Error bars are 61 sample standard error [solid symbols 5 proportion planulae
dead after 4 h in T . faulkneri extract; open symbols 5 proportion planulae dead after 4 h in solvent controls].
only 5. The pilot study showed that coral planulae were adversely affected by the extract in comparison to the solvent Fig. 1, Table 2.
A bulk batch of extract was prepared for further tests using a method that allowed the
Table 2 Pilot experiment: toxicity of Tubastraea faulkneri extract to planulae of Platygyra sinensis and Acropora
a
formosa Planula species
Source df
S.S. M.S.
F P
Platygyra sinensis Treatment
1 5.4
5.4 205.7
, 0.001 Concentration
4 2.1
0.5 20.4
, 0.001 Treat. 3 Conc.
4 1.5
0.4 14.1
, 0.001 Trend: Linear
1 0.4
0.4 17.1
0.000 Quadratic
1 0.8
0.8 30.0
0.000 Other
2 0.2
0.1 4.7
0.015 Error
40 1.0
0.0 Acropora formosa
Treatment 1
10.3 10.3
1422.1 , 0.001
Concentration 4
0.1 0.0
1.6 0.201
Treat. 3 Conc. 4
0.0 0.0
1.4 0.261
Error 40
0.3 0.0
a
Factor ‘Treatment’ refers to presence or absence of extract in test solution. Factor ‘Concentration’ refers to concentrations of extract and corresponding solvent controls. Shows significant P values , 0.05. For each
species, variances were homogeneous among treatment groups for interaction and main effects. Trends refer to partitioning of the significant interaction term using polynomial contrasts.
E .G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160
147
natural concentration to be calculated see Section 2.1. The number of experimental extract concentrations was reduced to allow more species of coral larvae to be tested.
Based on the pilot study, the other ten species of planulae were tested at three extract
21
concentrations of 50, 100 and 200 mg ml seawater. In addition, the bioassays using
Oxypora lacera and Platygyra daedalea were repeated at a later date with the addition
21
of four lower extract concentrations at 10, 20, 30 and 40 mg ml seawater.
The results for planula of each species were analyzed using two-way analysis of variance. The two main effects were ‘Treatment’ ethanol with or without Tubastraea
faulkneri extract and ‘Concentration’. The equality of variances associated with main effects and interactions was tested using the Brown–Forsythe test Brown and Forsythe,
1974; Keppel, 1991. If variances are heterogeneous, the probability of type I error is increased Keppel, 1991; Underwood, 1997 so marginally significant differences are
suspect. We guarded against this by reanalyzing such cases using a randomization procedure. This approach is based on the planulae being assigned to treatments at
random Manly, 1991. If the null hypothesis is true, then the treatments should have no effect. The pattern of mortality we observed would then represent one random
arrangement of the groups of planulae showing inherently variable mortality rates among the treatment combinations. The probability of obtaining this result by chance
can be estimated as follows. The observed mortalities can be randomly reassigned to treatment groups and a relevant statistic reflecting the difference in mortality among
groups is calculated for each rearrangement. Multiple randomizations are used to generate a frequency distribution for the statistic. The likelihood of obtaining the
observed value of the statistic or a more extreme one by chance can then be estimated. We used the software ‘RT’ Centre for Applications of Statistics and Mathematics,
University of Otago, New Zealand; the test statistic was the percentage of the total sum of squares associated with each factor. In each case, the test distribution was
conservatively based on 4999 randomizations plus the original sample Manly, 1991.
A significant interaction term would indicate that the mortality rates for planulae with and without extract diverged with changes in concentration. All interaction terms were
tested for linear and higher order trends with polynomial interaction contrasts for unequally spaced concentrations Keppel, 1991. If there was no significant interaction,
then a significant effect of ‘Treatment’ would indicate that mortality rates for planulae with and without extract were consistently different. In the absence of an interaction, a
significant effect of ‘Concentration’ would indicate that the solvent had an effect on larval mortality. The main effect of ‘Concentration’ was also tested for linear and
quadratic trends using polynomial contrasts for unequally spaced concentrations.
2.4. Isolation of pure compounds The active components of the methanol extract were isolated and purified by a
combination of chromatographic techniques directed by results from antimicrobial assays. The antimicrobial assays were described in detail in an earlier study Koh,
1997. Antimicrobial activity was used as a general indicator of bioactivity in fractions obtained at each step of the isolation process.
Coarse fractionation of the methanol extract was performed by flash column
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.G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160
chromatography on silica gel see Coll and Bowden, 1986. Briefly, the extract was dissolved in a 1:1 mixture of methanol and dichloromethane. A small amount of silica
gel was added to the solution and the solvents were removed using a rotary-evaporator. The dry, even mixture of the extract and silica gel was then loaded onto a silica gel
column for fractionation. Solvent mixtures were used to elute six fractions under vacuum, starting at 5 and finishing with 25 methanol in dichloromethane. The flash
column fraction with the highest antimicrobial activity was prepared for HPLC by loading it onto a C18 Sep Pak Waters Millipore Corporation and eluting it with
solvents of decreasing polarity. The first fraction was eluted with 4:6 methanol distilled water, the second with methanol, the third with 1:1 methanol ethyl acetate, the fourth
with ethyl acetate and the last with hexane. The first two fractions contained antimicrobial activity and were combined for reversed-phase HPLC no antimicrobial
activity was detected in the third and fourth fractions and nothing was eluted with hexane.
Reverse-phase HPLC was carried out on an Alltech Econosil C18 column 10 mm; 250 mm long and 10 mm internal diameter with an isocratic solvent regime nine parts
methanol and one part 5 ammonium acetate in distilled water at a flow-rate of 1.5 ml
21
min . Four major HPLC fractions were collected at the following retention times: 8,
9.5, 10.5 and 13 min. Ammonium acetate was removed from the fractions before they were tested for antimicrobial activity. The fractions were further purified under the same
HPLC conditions before they were characterized by melting point analysis, NMR, UV and mass spectrometry.
3. Results