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
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Bioactivity
of antarctic
References
sponge extracts(Z
TFRTFRTFR
LIy. rcum
TFR
TFR, BS
Cinachyra antarcticaTFR
TFR
Dendrilla membranosa-TFR
TFR
Gellius benedeni -TFR
BS
-BS
Gellius tenella*
TFR
TFR
Haliclona sp.
BS
Haliclona dancoiTFR,BS
Homaxonella balfourensis-TEA
TER
TFA
Inflate/la be/li
TEA, BS
lsodictya erinaceaBS
Isodictya setifera' -BS
TFR
TFR
TER
TER
BS
BS
Kirkpatrickia variolosa-TEA
-,
TER,BS,EC
BS
Latrunculia apicalis
TEA,BS,EC
BS
Leucetta leptorhapsisEC
Mycale acerata
--TFA
BS
Polymastia invaginataTFR,BS
-TFR
Rose/la nuda
BS
Rose/la racovitzaeTEA
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.
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