Table Of ContentPRODUCTION OF BIOACTJVE FURANOSESTERTERPEN£ TETRQNIC ACIDS \s
POSSiBLE INTERNAL HEMICAL DEFENSE MECHANISM IN THE SPONG1
(
IRCINIAFEUX(PQJUWERA: DEMOSPQNCHAE)
SVFN 2EA, FFRNANDOJ. PARRA, Al IJANDRO M \RTINT7 ANP CARMFNZA DlQVt
Zea, Y. r'arr-a. u.. Manfoez, v & Duque, C, 1999 (><s JO: Production oj bioaclive
furanoscsterterpene tetronic acids as possible interna! chemical defense mechanism in the
sponge bvirtia felL\ i Porifent Demospongiae). Memoin oj the Qui n ..-./ Museum 44:
696, Rnsbane. ISSN 0079-8835;
Marine sponges trf the genus Jrcittia fPorifera, Demospongiae, (iciniidae) are known lo
produce several linear fuumosesterpenetetronic acids<I- lAsi ivith antimicrobial, cytotoxic
andamilumoralproperties.Irciniafeiix isacommonandabundantsponge fromSantaMarta,
CokxnbranCaribbeanSea,comainiugPTAsinquantiti& upto4.5% cri '-.n-treedry ti
\seight.FTAconcentrationwasquantifiedbyIIPI Ca(let organicextractioninindividualsof
ux The following results wen. obtained: 1 )peripheral tissues had greaterconcentration
tlohatnheintaemrbniaelnttissiulelsu;m2i)nattoitoanlbwohde>rePTiAndciovnicdeuanltsratliivoend (in rielation 10- nddeptthg,niafnicdanctolymrpelated
: |i i nousshaded vs. open loliglu,and localitiesdiffering inwater turbidity); 3)there \\a.
significant variation in FTA concentration throughout the time of stud} ( Iune-December
i|;4)overa2 monthperiod it was found thai experimental shading induced a signilfc
ipcrease in total body F IA concentration: 5> there wasa strong F1A increase (inascale df 1
k-2 months) when sponges were manipulated in depth Ira cperiroeuts and
when the> were purposely injured and; 6) intact or injured individuals did not exude
measurable quantities of FTAs into the surrounding medium in laboratoiy conditions,
logether, these results indicate rhat FTAs have seme adaptive \aluc. but probably not in
mediating external ecological interactions, but instead acting as allamonal internal
suppressorsand'orantibiotics The shads.-dependent production ofFTAs suggest that thi
substances may prevent parasitisatiun by photosynthetic Aphanrna;- WrtHype
endosymhionts, when the ambient illumination is below their compensation point.
Additionally,asthespongebecomes nunc bete rtrophic underlowerlight levelsitmay have
I
anincreasedneed forantibioticsinthechoanosometopreventbacterial food Irom becoming
infectious. Finally, dUtif) Utnd healing, increased FTA levels nun1 also act as internal
O
antibiotic protection. Ptirifem, ficmnoazsterierpene tetroiiic acids irc'mta, chemical
Interna!defense, < 'aHbbean
, nail szeau.- A Fernanda .I. Pan-a Cniverskiud Xueionalde
Coiomhia (DeportamentQ de Biotagitx}, Institute de lawstigurtontt Harinas y Cosletm
'Jose Bcnno lives deAndrei*'. /Xl'EMAR. AA I0-K), Santa Marta, Colombia; Alejandro
Martinez. haeultuJ de Ouinttca Far/neeetihxa >' n vert fad A Unioqtiia. A4 1226,
MedeU'tn Coiomhia Carmenza Dtujtie Depatiamenfn de QuimiCQ, (niversidad \aeionul
de€olomb\ 100690. Bogota, Colombia; 17March 1999.
i I I
The search for new drugs has led to the epibiosis or external damage by roaming
discovery of a variety of bioaetive secondary browsers (e.g. Thompson. 19X5; Walker cl al.,
metabolites in many terrestrial and aquatic 19S5; ThompsonelaF, I9S7; Davis, 1991 ). Intra-
organisms (Carson, 1994). ltt themarine realm. Specific variation in toxicity 0V in secondary
however, little isknown about the use thatsource metabolite composition has been documented in
organisms make of these substances- It is several groups ofbenthic sessile; organisms (see
commonly thought thai sessile organisms use re\icw in Bee'crro et aF, 1995). In sponges there
bioaclive secondary metabolites as signals to area few casesinwinch intraspecific variation of
communicatewithconspccillcs. deterpredators hioaetive secondarymetabolites, bothintypeand
tli
o\^ adults (e.g. Rakus et al.. I9S6: Pawlik et aF, concentration, has been documented (Thompson
L99S; WullY, 1994; 1-995), r>r propagules et al., 198?, 1985, 1987; Kreuter et al. 1992'
(Thompson et aF, 1983), to actively or defens- BecerrO el al.. 1995). This variation has been
ively compete tor space [e.g. Sullivan et aF. attributed to individual physiological defensive
1983; Acrts & Van Soest, 1997F or to prevent responses to differential env'ironmental
9 A
688 MEMOIRS OF THE QUEENSLAND MUSEUM
pressures within its habitat range, especially the natural and experimental conditions. We found
degree of spatial competitive interactions with an inverse relationship betvvecu FT
neighboring macrobiota, and epibiosis concentrationandambientillumination,agreater
prevention(Thompsonetal., 1987;Becerroetal., concentration ofFTAs in internal tissues, and an
1995). Intra-individual(intercellular)variationin induced production of FTAs in wounded
the location ofbioactive secondary' metabolites sponges. Here, we report on, and interpret these
has been documented in a few sponges. In two results in terms ofinternal defense mechanisms.
eases, bioactive metabolites were found in
inclusionswithin spherulous cells, located mostly MATERIALS AMD METHODS
nearthesurfaceofthesponge oraroundexhalant
STUDY AREA AND SOURCE ORGANISM.
canals. These cells appear to disintegrate and &
releasetheirinclusions,resultingintheexudation Irciniafelix (Duchassaing Michelotti, 1864)
ofmetabolites through the pinacoderm, into the was collected from rocky shores and mid-depth
excurrentcanals,andthenintotheboundary layer fringingreefsofPuntadeBetin,andadjacentport
aroundthesponge.Thesemetabolitesarethought dock-pilinas in the bay of Santa Marta city
to be used forexternal defense-offense, but they (iri5'N, 74°13,W), and in rocky shores and
may also be released into the mesohyl matrix for fringinrg reel's of Isla Aguja, further to the NE
internal use (Thompson et al., 1983; Uriz et al., (11°1 N,74°12'W),ColombianCaribbeanSea.
1996b). ComparedtoIslaAguja, PuntadeBetingenerally
hasmoreturbid waters, and is subjectedtogreater
Species of Ircinia (Demospongiae, Dictyo- sedimentation loads from an adjacent river, the
ceratida, Irciniidae* are known to produce linear city sewage outflows and commercial port
furanosesterterpenes (Cimino et al., 1972a, b; activities. Reefcorals in this locality have also
Lumsdon et ah, 1992; Urban & Capon, 1992; suffered greater mortality, and reefs are amply
Caponeta!., 1994; Davis&Capon, 1994;Murray colonised by sponges (Zea, 1994), especially by
et al., 1995). Recently we reported on three species ofIrcinia (Parra, 1997). Irciniafelix has
Caribbean species ofIrcinia(Lfelix, I. strobitina been described fromthis locality in detail by Zea
and/. campana), containing the novel (7E, 12E, (1987),andreferencematerial isdeposited in the
18R, 20Z)-variabilin as themajor (58%-59.8%) collections ofINVEMAR, Santa Marta, and the
furanosestertcrpene tetronic acid, followed by a instituto de Ciencias Naturales, Universidad
mixture of(8E, 13Z, 18R, 20Z)-strobilinin plus NacionaldeColombia,Bogota. Intheinvestigated
(7E, 13Z, 18R, 20Z)-felixinin (27.1%-28.6%) localities this species lives in densities from 4-50
and a mixture of the new compounds individuals/40m"; it is usually thickly encrusting
(8Z,13Z,18R,20Z)-strobilinin and (7Z,13Z,18R, to cushion-shaped, occupying areas from about
20Z)-feiixinin (13.1%-13.9%) (Martinez et al., 30-100cm", and having maximal thickness from
1995b, 1997). The greatest concentration of about l-6cm; surface is conulose, usually clean
FTAs occur in /. felix, followed by /. campana and free ofepibionts, with several interspersed
and /. strobilina (Martinez, 1996). These FTAs oscules, 3-5mmdiameter, slightly raised by a
were also found to occuras branched chain fatty membranous collar; external color in life varies
acid esters, a unique combinationneverreported from shades ofmaroon and amber in specimens
before in nature (Martinez et al., 1995a). FTAs in well illuminated locations, to dirty cream in
have been demonstrated to have a variety of shaded ordeep locations; internal color is cream
pharmacological properties: e.g. antibiotic (Parra, 1997). Below a sand-filled ectosomal
(Faulkner, 1973; Martinez, 1996), cytotoxic reticulation (cortex), there is a layer with dense
(Martinez, 1996), antimicrobial and antitumoral aggregations of cyanobacteria of the
(Gamboa & Pinzon, 1997), analgesic and anti- Aphanocapsafeldmani-type, whose pigments are
&
inflammatory (Del Valle Vargas, 1997), responsible for the color ofthe sponge (Rutzler,
calcium transport inhibition (Beveridge et al., 1990; Vicente, 1990). This species is typical for
1995). Pawliketal.(1995), arguedthatstructurally thegenus in beingvery tough anddifficultto cut
complexsecondarymetabolites, whichare usually or tear largely due to a dense reinforcement of
presentathigh concentrations in sponges, canbe spongin fibrils throughout the mesohyl. Species
physiologically expensive to produce, and thus ofIrcinia alsoyield a characteristic sulfur-garlic
must have an adaptive purpose. To test whether stench when handled (Bergquist, 1978), releasing
FTAsplayanecologicalroleinIrciniawestudied severalsulfi.traridcyanidevolatiles(Bonilla, 1997).
the intraspecillc and intra-individual variationin Two morphotypes are readily distinguishable in
FTA concentration in /. felix. under various SantaMartapopulationsofI.felix: 1)encrusting,
CHEMICAL DEFENSE IN IRC1NIA FELIX 689
dark amber surface, oscular skin collar dark pooled for further analyses and calculated as mg
brown, and,; 2) encrusting to cushion-shaped, FTA g"1 ash-free dry weight ofsponge.
maroon surface, oscular skin collar white (Zea,
1987). Only the latter morphotype, which is the FTA CONCENTRATION IN 1RC1NIA FELIX.
mostabundantinthestudyarea(Parra, 1997)was Depth and ambient illumination factors. To
used for the chemical ecology studies presented initially explore ifthere was variation in natural
here. variabilin concentration in tissues between
individuals across various environmental
EXTRACTION AND QUANTIFICATION OF conditions,twospecimensofl.felixwerecollected
FTAs. Whole sponges or fragments were inJune 1995 ateach offourdepths (5, 10,15 and
immediately frozen upon return to the field 20m), in conditions ofopen exposureto ambient
laboratorybase(INVEMAR, SantaMarta). Frozen illumination at the rocky shore and fringing reef
material was air-shipped to theNatural Products ofPuntade Betin, and twomoreat4-9m depth in
Laboratory of the Universidad Nacional de the adjacent well-shaded pilings of the Santa
Colombia at Bogota, for chemical analyses. Marta port dock. Statistical differences in
Extraction and quantification ofbioactive FTAs variabilin concentration between depths and in
wasdeveloped by Martinez ( 1996)and Martinez pilings were tested by one-way ANOVA;
et al. (1997), and standardised for this study as variabilin concentration in relation to depth was
follows: 5-10g o\" wet sponge were cut and investigated by regression analysis. For all
macerated first inmethanol (MeOH) and then in statisticaltests,includingthosementionedbelow.
ethyl acetate (EtOAc), each for 15mins, data was tested for homogeneity of variances
removing the supernatant by filtration aftereach between treatment combinations (Bartlett test),
solvent addition. Each supernatant was and for normality of residuals (Kolmogorov-
separately vacuum-dried at 35°C in a rotatory Smirnov test); when suitable, transformations
evaporator, then diluted in EtOAc, mixed, and wereapplied andmeansand standarderrorsback-
partitioned repeatedly with H2 to eliminate sea- transformed for presentation (Sokal & Rohlf,
water salts. The EtOAc fraction was collected, 1981).
dried with anhydrous sodium sulfate, filtered,
Variabilin concentrations in peripheral vs.
vacuum-dried and weighed. Ash-free dry weight
of the solid sponge residual was obtained by choanosomal tissues. To compare variabilin and
total FTA concentration in peripheral tissues
subtracting from the oven-dry weight (at 1 15°C) (including pinacoderm and peripheral
the ash weight obtained after combustion in a mm
muffle furnace at 400°C. To prolong its stability choanosomal tissues a few below the ecto-
during storage, the extractwasthenacetylated in some) vs. internal tissues (deeper within the
a 20ml mixture of acetic anhydride-pyridine choanosome), in open vs. shaded locations, two
(1:1). Theacetylated FTAswerethenpurifiedby specimens werecollectedat 6-7m in thePuntade
silica-gel column chromatography, dried and Betin rocky shore inJune 1995 (variabilin only),
andtwomoreat5-6mintheadjacentdockpilings
stored under nitrogen atmosphere at 0°C until
use. Acetylated FTAs were subjected to IIPLC in September 1995 (variabilin and total FTA).
for final purification using MeOH-HjO (85:15) Peripheral tissues were dissected upon return to
the laboratory and stored and processed
as mobile phase ataflow rate of1ml min~ and a
sCatpacteilolnaPraykpChal8se(,25m0oxn4i6tmomrini.gd.)atco2lu7m0nnma.s dsiefpfaerraetnecleysfirnomvacrihaobainlionsoamnadlFtTisAsuceso.ncSetnattrisattiicoanl
between tissues were compared by one-way
Chromatograms typically gave three peaks ANOVA
between 9 and 12mins, whose subtractions wrere foreach habitat separately.
known to contain five different acetylated FTAs Exudation. Two assays were carried out to test
(Martinez et al., 1997). Since the largest and whether FTAs are released by undisturbed and
latest subtraction contained pure (7E, 12E, 18R, wounded l.felix. 1) Nine darkened and aerated
20Z)-variabilin acetate (henceforth refered to as aquariafilledwith0.5Loffilteredsea-waterwere
variabilin),a lOu-guT1 solutionofthiscompound set up in the laboratory. Six specimens ofl.felix
previously obtained was used to construct a were carefully collected with the substratum at
calibration curve to calculate sample concen- 10m depth in Punta de Betin in October 1995
trations. Initialquantificationsweredoneonlyon using hammer and chisel, and each placed in an
variabilis but as the structure ofall FTAs were aquarium. Three specimens were deeply
being elucidated and found to be bioactive wounded with a razor blade whereas the other
(Martinez, 19%), data for the three peaks were three were left undisturbed; the three aquaria
690 MEMOIRS OF THE QUEENSLAND MUSEUM
without sponges were used as controls. each depth), both appropriate for unbalanced
Unwoundedspecimens were checked forvitality designs(SAS Institute Inc., 1988). Timechanges
by observing the pumping of water through were tested, comparing in a separated one-way
oscules. After 4 hours, sponges were removed ANOVA, samples from Puntade Betin collected
and the water stored in cold (4°C), while it was in June (see above). Septemberand December.
beingvacuum-filteredthrough RP-8cartridgesto Wound-induced production. To test for FTA
retain organies. Cartridges were then kept frozen production in tissues after wounding, six speci-
isnimtihleardamrakn.ne2)rTthoitsheasfsiarsyt,wiansNcoavrerimebdeorut19i9n5.a mPuenntsawdeereBleotcianteidnaNnodvteamgbgeedrat191905m. dIenipttihallfyr,oma
with a single specimen for each treatment. The wound was produced by cutting free from (he
waterwas immediately partitionedin EtOAc,the edgea2-3cm widefragmentfrom eachspecimen,
organic fraction dried, put under nitrogen which was immediately frozen to measure initial,
atmosphere and stored frozen in the dark. After natural FTA levels. A similarly-sized fragment
sMheipOmHentantdo BEotgoOtAac, ctaortrreildegaessewoerrgeanfileusshaenddwitthhe wwoausnda,gaifnromtaekeanc,h ocfuttthirnege spapreaclilemlentsostehveenindiatiyasl
combined extracts dried. Extracts from both later, and from each ofthe otherthree specimens
experiments were aeetylated and quantified as 14 days later; these were frozen to measure FTA
mentioned above. concentration changes in the wound area in the
Ambient- and time-related differences, and interveningperiodasaresultoftheinitial wound.
experimentally inducedproduction. To compare Initial and final FTA concentrations were
total FTA concentration in localities having compared (breachtimeinterval set by a t-student
different environmental conditions, three test. FTA changes for individual sponges were
specimensofLfelixwerecollectedateachoftwo compared (null hypothesis of no change) by a
depths (10 and 20m) in Punta de Betin and Isla paired t-student test, separately for each time
Aguja, in September 1 995. Three more interval.
specimens were collected at each of the same
depths in Punta de Betin in December 1995. RESULTS
Simultaneously, shadinganddepth transference
experiments were carried out at Punta de Betin NATURALFTALEVELS.Totalbodycontentof
from September to December 1995 to test for FTAs in I. felixranged from about l-46mg g"1 of
additional production of FTAs. The above- ash-free dry tissue weight (Table 1; 0.1%-4.6%
mentioned specimens served as initial and final by weight). Significant variation in total FTA
controls for natural FTA levels. At each of the and/or variabilin (58-60% of total) content in
two depths, three individuals were shaded by a samples was found as follows.
canopyofwirenailed tothecorallinebottom and Ambient illumination factor. There was a
covered by a black soft polyethylene plastic. As significant increaseinvariabilinconcentrationin
controls, two individuals were also covered by a tissues of /. felix with increased depth at the
canopy but withtransparent plastic. Also, ateach fringingreefofPuntade Betin inJune 1995 (Fig.
depth, a PVC tube frame holding four, 5cm-wide 1). This trend followed a potential regression
Plexiglas beds was nailed to the bottom. Four model [variabilin] = 0.0453 Z1,82 RML95,
specimens at each depth were carefully collected 0.0KPO.025 (see Martinez, 1996).,Since light
together with the substratum using hammer and intensity decreases exponentially with depth in
chisel, and fixed tightly on each bed with plastic sea-water, ambient illumination (not measured)
cable lies; in each frame, two specimens came was assumed to at least relate partly tovariabilin
from the samedepth as controls formanipulative concentration. Deviations from regression,
experiments,andtwospecimensweretransferred however, accounted for a significant part ofthe
from the reciprocal depth. All sponges were model (R"=0.04, 0.025<P<0.05), denoting that
collectedsimultaneouslyinDecember 1995, frozen other factors apart from depth (and light) are
and processed.
importantindeterminingvariabilinconcentration.
Natural,total FTA levels fortwo localities, two In addition, variabilin concentrations similar to
times (initial and final), and under shading and those found at 20m, were measured in sponges
transference treatmentsandcontrols werestatist- collected simultaneously at 4-9m in the shaded
ically compared against depth in a two-way nearbypilingsofSantaMartaport(Fig. 1). These
ANOVA (type III sums ofsquares), and a Tukey valuesweresignificantlygreaterthanthoseofthe
multiple comparisons procedure (separately for spongescollectedat 5m intheopen fringingreef.
CHEMICAL DEFENSE IN IRCINIA FELIX 69]
20 Further comparisons oftotal FTA concentration
in September and December 1995 at Punta de
A Betin revealed a similar depth trend (Fig. 3A,D,
15- A representing 10 and 20m depths, respectively).
The same was also true for Isla Aguja, although
b) h theabsolute levelsweremuch lowerthatatPunta
E de Betin at both depths, presumably due to
I 10 < greater light penetration in its generally more
transparent waters. Together, these results show-
>ra B B that ambient illumination is an important factor
5 ; relatedto FTA natural concentration intissuesof
B r=-l Lfelix.
Timefactor. At 5m depth, both in open (Puntade
. Betin rocky shore) and shaded (dock pilings)
4-9m 5m 10m 15m 20m locations, there was no significant difference in
Shaded Open variabilin concentration between samples
Lccatian collected in June and September (two-way
ANOVA, R2 [time] =0.007, P= 0.48, for data,
FIG. I.Concentrationof(7E,12E,18R,20Z)-variabilin compare Fig. I and Fig. 2 choanosomal tissue).
(mg g-1 ash-free dry weight of sponge, mearufcone Similarly, for 10 and 20m depths, there were no
standard error) in Ircinia felix under natural significant differences in variabilin
conditions, inshaded(commercial portdock pilings, concentrationbetweenspongescollectedinJune,
4-9m depth), and open (rocky-shore and fringing SeptemberandDecember 1995 atPunta de Betin
reef,5-20mdepth)locationsatPuntadeBetininJune (two-way ANOVA on time and depth, R" [time]
1995. Bars sharing the same letter are not
significantly different from each other in a Tukey =0.01, P=0.33, data from Fig. 1, and corresp-
multiple comparison procedure (after one-way onding variabilin values ofFig. 3A, not shown),
ANOVA, R2=0.93, P=0.004, n=2 samples per norwasthereanydifferenceintotalFTAconcen-
location-depth). tration between September and December (see
Fig. 3A).
15 T Variabilininspongetissues. Peripheraltissuesof
I.felixshowedasignificantlylowerconcentration
ofFTAthan internal tissues, both in shaded(dock
pilings, one-way ANOVA, R2=0.96, P=0.0002,
n=2) and open locations (Punta de Betin rocky
shore,one-wayANOVA,R2=0.99,P=0.003,n=2)
at similar depths, although concentrations were
significantly higher in shaded sponges in both
tissues(approximately23 timesinperipheraland
12 times in choanosomal tissues to that of
sponges open locations) (Fig. 2). This implies
that lower FTA concentrations in peripheral
tissuesisnotdirectlyduetoambientillumination.
FTA PRODUCTION INDUCTION. Exudation.
Waters surrounding individuals ofI.felix kept in
aquariaforafewhours, evenwhenwounded,did
not show traces of FTAs; both RP-8 cartridge
Open Shaded filtration and direct EtOAc extraction gave
Location negative results. Hence, it seems that FTAs are
not released into the surrounding water of the
FIG. 2. Concentration of(7E,2E,18R,20Z)-variabilin sponge, at least in detectable quantities.
(mg g~' ash-free dry weight of sponge, mean±one
Shade-inducedproduction. Shadingexperiments
ostfaInrdcairndiaerfreloirx)iinntpewroipdhiefrfaelrevnst.acmhboiaennotsoilmlaulmitniastsiuoens at 1 and20mdepthsinPuntadeBetinrevealeda
regimes (open rocky shore, 6-7m depth, June 1995; significantly greater FTA concentration when
shaded dock pilings, 5m depth, Sept. 1995). sponges were covered for 3 months with a dark
—
i
692 MEMOIRS OF THE QUEENSLAND MUSEUM
so- „ 50T 9 times, t=18.19, P=0.003, n=3) after
40- A * : wounding sponges at 10m depth in
30- Punta de Betin fringing reef(Fig. 4).
20- Initial levels were similar forthe two
10- B 6 3 10
r~ K sets of three sponges (t-student test,
f). i 1 1 1 1
t=1.08, P=0.34). Throughout the
Locality-Month Locality-Momh experiment, the wound area was
Naturalconditions Naturalconditions
50-r c bleached, and a substantial release of
mucus was noted while the wound
I 30- B~, was sealed.
t 20- Transference oXdepth experiments
!«:
(see above, Fig. 3C, F) elicited FTA
0-
Transp Dai Transp. Dark increases due to manipulation, prob-
Shadingtreatment Shadingtreatment ably also as a result of internal and
5400- D CD c „a> 5400-- c c F esxqtuereneazlidnagmagtehdemdonetitgohstpotnogestbhye
30- I 30- experimental frame. A few days after
20J t 20- transference, partial bleaching and
10- f 10'. discoloration was noted; a few
n- Same , 20m 1 o-l Same 1 1 m I specimens were necrotic in areas
Depthtransference Depthtransference contactto the cable ties. Slight differ-
ences between transference sets were
FIG. 3. Tolal FTA concentration (mean^onestandard error, mgg"1 probably due to the degree of
ash-free dry weight ofsponge, mean±one standard error) at 10m manipulation, which elicited more-or-
(A-C)and20m(D-F)depthinIrciniafelixundernaturalconditions iess damage.
(A and D) at IslaAguja(IA-September 1995) and Puntade Betin
(PB-September and December 1995) fringing reefs, and after DISCUSSION
shading(BandE)anddepthtransferenceexperiments(CandF)at
PuntadeBetin. Barsoneachdepthhavingthesameletterwerenot Thisworkisthe firstdemonstration
significantly different from each other in a Tukey multiple of intraspecific and between-tissue
comparison procedure (after two-way ANOVA, Logl0 variationinFTAcontentsinthemarine
transformeddata, means and standard errors backtransformed, R3 sponge Ircinia felix, in relation to
full model =0.99, P=0.000K n=3 for each treatment-depth several environmental factors and
combinationexceptforshadingtransparent(Transp.)controlsand biological stress conditions. A slight
depth transference, inwhich n = 2). degree of variation in deterrence of
feedingbyfishwasfoundinpelletised
shade, than when sponges were covered with extractsfromCaribbeanIrciniaspecies(including
transparent plastic, or were left alone under I.felix),indicatingindirectlythattoxicsecondary
natural light. Initial and final natural levels, as metabolitesmayvaryintraspecifically(Pawliket
wellastransparentcovercontrols,weresimilarin al., 1995). Our results show that FTAs are not
FTAconcentrationatboth depths,whilethe dark exuded free, in mucus, or after injuries, into the
shade elicited a 5 times increase at 10m, and an surrounding water, and that they also occur in a
about2timesincreaseat20mdepth(Fig.3B,E). lower concentration in the peripheral tissues.
In contrast, further experiments failed to Hence, an external defensive (epibiosis, pred-
demonstrate an expected decrease in FTA ation) and offensive (competition for space) role
concentrationwhen spongesweretransferred from cannot be directly ascribed to FTAs. This
20m to 10m depth, due to a manipulation effect. contrastswiththefactthatIrciniaspeciesusually
Both control andreciprocal transferenceresulted have an epibiont-free surface(Parra, 1997), their
in a significant increase after three months to a pelletised crude extracts are not consumed by
similar FTA concentration, when compared to fish (Pawlik et al., 1995), and they have been
untreated controls, regardless ofdepth (Fig. 3C, found to be aggressive in situ against corals
F). (Aerts& Van Soest, 1997), andthesedefensive--
Wound-induced production. A significant offensivemechanismsarethoughttobechemically
increase in FTA concentration from the initial, mediated. PerhapsFTAsarebad-tasting,ortoxic if
natural levels were found seven days (x 6 times, swallowed,hencetheabove-mentionedrejection
t-studenttestt=4.74,P=0.04,n=3)and 14days(x by fish (J. Pawlik, pers. comm., 1998).
CHEMICAL DEFENSE IN IRCINIA FELIX 69:
Nevertheless, otherinvestigations carried out by a natural function to avarol in lieu of the
us showed that /. felix passively and actively possibility ofit being a by-product ofextraction
(upon injury) releases into the surrounding and manipulation procedures. In addition, there
medium other noxious volatile compounds are no reports of environmentally induced
(Bonilla. 1997)which may be partly responsible variationinavarol.AsforFTAs,themildextract-
fortheabove-mentionedecologicalinteractions. ion and purification procedures used are not
Our results are also in contrast with those strong enough to either generate their functional
reportedforotherspongesthathavesofarshown groups, or, in case thesemolecules only occurin
environmentally induced variation of bioactive the sponge tissues as fatty acid esters (cf.
secondary metabolites, in which the higher Martinez et al., 1995a), to breaktheir ester bond
concentrations of these metabolites have been (Martinez, 1996). Hence, we argue for an
found in the periphery ofthe sponge, or in cell adaptive, internal defense functionofFTAs based
types surroundingthe internal canal system (e.g. on: 1) their presence in the tissues in free, bio-
Aplysina fistularis, Rhopaloeides odorahile, active form,andin relativelyhighconcentrations,
Verongia aerophoba, Crambe crambe; see implying an energetic cost ofproduction which
Thompsonetal, 1983, 1987;KreuteretaI., 1992; shouldbebalancedagainstotherneeds(cf.Pawlik
Uriz et al., 1996a; respectively). Some ofthese etal., 1995); 2)natural (between individuals and
sponges contain metabolites which may be between tissue areas of the body) and
released into the milieu, either in areas ofdirect experimentally-induced variation inconcentration,
contact with other sessile organisms, or exuded inrelationtoambientillumination;and3)injury-
into the water (inside the canals or into the -inducedproduction.
surrounding medium), to act in various One could also argue that the natural and
chemically-mediated interactions (Sullivan et al., shade-induced variations found in FTA concen-
1983; Thompson et al., 1983; Thompson, 1985; trationareanartifactoftheirlabilitytolight,orto
Walkeret al., 1985; Kreuteretal., 1992; Becerro the result of a light-dependent biosynthetic
etal., 1995). mechanism (e.g. Kreuter et al., 1992). However,
However, similar to FTAs, the bioactive the relatively low FTA levels in choanosomal
compound avarol and its derivatives, produced tissues in sponges located in open conditions
by the Mediterranean sponge Dysideaavara, are cannot be ascribed to a light-dependent effect
found only in the choanosome, specifically because the peripheral tissues have a strong
within choanocytes (Uriz et al., 1996b). In fact, pigmentation (Parra, 1997), that probablyprevents
the latterauthorshavecautionedagainstascribing light from reaching deeply into the choanosome
&
(Wilkinson Vacelet, 1979; Wilkinson, 1980).
Similarly, finding a lower FTA concentration in
sc
peripheral vs. choanosomal tissues in discolored
sponges from very dark habitats argue against
50.:
light-dependent degradation.
The following are possible (and not mutually
;oi 401 exclusive) explanations for our results in regard
E I to internal defense.
< 30.
FTACONCENTRATIONANDPRODUCTION
INDUCTION IN RELATION TO AMBIENT
ILLUMINATION. The causality of light (and
tissue)-relatedFTAnaturallevelsandshade-related
10 l production may be interpreted in the following
ways.
I) FTAs are used for (partial) control of
Initial 7d Initial 14d Aphanocapsafeldmani-type cyanobacterial symb-
Timeafterwound ionts. Apart from cellular mechanisms for
repression (e.g. phagocytosis) ofendosymbiont
FIG. 4. Changes in total FTA concentration (mg g~' population growth (Simpson, 1984), anti-
ash-free dry weight ofsponge, mean±one standard microbial secondary metabolites could help in
error)infrciniafelix,1d(openbars)and 14d(shaded their repression. Assuming an antibiotic or
bars)afterelicitinga wound; n =3 foreach set. cytostatic affect ofFTAs ox\A. feldmani, a lower
694 MEMOIRS OF THE QUEENSLAND MUSEUM
FTA concentration is retained in the ectosomal contrast,otherstudiedspongeshaveshownannual
tissuesofLfelix, wherethisbacteriais located, to cyclesoftoxinproductionrelatedtointernalphysio-
allow it to carry out its symbiotic role. Also, an logical cycles (e.g. reproduction), and external
overall concentration of FTAs is maintained in ecological pressures (e.g. competition forspace)
inverse relation to light levels, to prevent A. (Turonetal., 1996).
feldmani from parasitising the sponge once its FTA PRODUCTION INDUCTION AS A
production/respiration ratio is below unity (i.e.
belowthecompensationpoint).Thisroleisdiffkult RESULT OF INJURIES. FTAs may act as a
to test from our observations under natural mid-term (days to weeks) internal antibiotic
conditions, since light itself may be directly protectionduringwoundhealing(e.g.afterpredator
responsible For A, feldmani control. In fact, in bites,sandscouring,etc.).However,thisincrease
contrastto other,morephototrophic sponges(e.g. does not lead to exudation. Instead, rapid
Seddon et al., 1992) and zooxanthellate corals (minutes to hours) release of noxious volatile
(e.g. Titlyanov, 1981; Jaubert, 1981), in which compounds (Bonilla, 1997) may act in the short
symbiont density and total chlorophyll-a term to protect wounded sponges ofthe genus
concentration tend to be inversely related to Ireinia, as it has been found for other sponaes
ambientillumination(andhencedepth),inI.felix (Thompson, 1985; Walkeretal., 1985).
coloration intensity (and also chlorophyll-a Regardless ofits specific mechanism ofuse, it
concentration) decreases with decreasing illum- can be advanced that FTAs possess allomonal
&
ination (unpublished results). Through cell effects (cf. Whitlaker Fenny, 1977), acting as
dissociation procedures, we have failed to do internal supressors and/or antibiotics against the
precise direct counts ofA.feldmani from I.felix sponge's own bacterial endosymbionts, or
collected at various depths and light regimes. against external bacterial invaders from food or
because the dense network of spongin fibrils through injuries.
prevented a total separation of bacterial cells
(histological procedures are needed). In short, to ACKNOWLEDGMENTS
test this role, the sponge-cyanobacterial
metabolism in relation to light, and the capacity This research was funded by COLCTENC1AS
ofthiscyanobacteriato liveheterotrophicallyoff (Grants 1101-09-039-90, 101-09-129-95,
the sponge, should be explored, as well as a contract 228-95), the Universidad Nacional de
direct, in vitro control of population growth by Colombia, and the European Economic
FTAs. Wehavesucceededin isolatingthecyano- Community (CI1*. 0448.C |JR]). INVEMAR is
bacteria, but have failed in its culture; trials are thanked for its logistical support. These results
under way to test the effect of crude sponge arepartofthesespresentedaspartial requisitefor
extracts on recently isolated cyanobacteria. thedoctoral degree inChemistry (A.M.), andthe
professional degree in Biology (F.P.),
2) FTAs are used to help in controlling bacterial Universidad Nacional de Colombia.
food (e.g. Bergquist & Bedford, 1978). Contribution No. 617 of Instituto de Investig-
Assuming I.felix turns more heterotrophic with acionesMarinasyCosteras "JoseBenitoVivesde
decreasing light (as translocation of pholo- Andreis' - INVEMAR, and No. 152 ofMarine
Biology Graduate Program, Universidad
synthates from cyanobacteria decreases), there
may be an increased need for antibiotics in the Nacional de Colombia, Faculty ofSciences.
choanosometopreventbacterial foodfrom parasit-
isingthe sponge. The sponge may also make use LITERATURE CITED
ofits own stores ofendosymbiotic heterotrophic ARRTS, L. & SOEST, R.W.M. VAN 1997.
bacteria as food when photosynthesis is off Quantification of sponge/coral interactions in a
(Simpson, 1984; but see Wilkinson et al., 1984), physically stressed reef community, NE
and may use FTAs to resulate its bacterial flora Colombia. Marine Ecology Progress Series 148:
(Thompson et al., 1983). 125-134.
In the above context, the relative constancy of BAKUCSh,emCi,caTlAReGcoElToTg,y No.fMm.a&rinSeHUoLrTgaEn,isBm.s:198A6n.
FTAconcentrationinLfetixthroughoutthestudy overview. Journal of Chemical Ecology 12(5):
period may indicate that seasonal changes in 951-986.
ambient illumination and degree ofphotolrophy- BECERRO, M., TURON, X. & UR1Z, MJ. 1995.
heterotrophy arenotenoughto exertan important Natural variationoftoxicity inencrustingsponge
effect on the Aphanocapsa-FTA system. In Crambecrarnbe(Schmidt) in relationtosizeand
1
MICALDEI
F 6*>5
environment,JoumaioK hemicalEcolq processestoadapt tolighl variations. Pp. 55-58,In
i of the 4tn Internal onal
BERQl '1ST. P.R. IWS.Sponues 11Jutcbinson:Lundon). Symposium- Vol. 2. [University oPPhilippines:
BERQUIS'I; PR A: BEDFORD. .U. I97S. The Manila).
incidence of the antibacterial actrvtrj fn mi rim K.REUTER, M., ROBITZKI A., CHANG, S
eDreamtoiosnpsonM:ariin-e Biloelmoal©ic4a:n2d)5g-e2o2g1r,aphic'consid- sHuACl'lM* MrsANR.N,.M1CMH.,AEISCiIs,1MR.O.PKFlRJ,AJMII. .V
BEVEJtlDCE, a.a., nil UMi ANDERSON, A MUI I.KR„ \V. I9(J2 Production oftfiecytostatic
CAPON, R.J. IU95. The efleel ofOre marine ayenl aeroplysinm by the spOJ
natural product variabilis on con.. . ictivirj hub m w vitm caii' iparati
of ntimiuemcgautiinoena-pig i"l-e|u3m5.. Phunnuccui ii I LI MSi DONe,m IiXt.r CrAidPPOhNys,iolUo.g,yT1H01O(1M)A;S1,83-S., &
BONtLLA, \ l'>S>7. Carueteri BEVERIDGE A, 1992. A new sesterlerpene
necstprovnijdaad maanrliimniacroTbricanMaade/W/l\os \B.ilSatclli Thdeesilsa an Australi1 an lmaapreinnetprsepnoynlagteed/)*qUino|aI
I i.eivosM> i. (UoiversM d Australian Journal ofChemistry 45; 1 121-J32S,
Naeional de Colombia, Facultad dc Ciencias: mxriimv, \. 1996. Esnidioquh
Bogol 1 igica dc tsponjas marinas colombumas del
CAPON."R.. DARGAVILLE. 1, & DAVIS, R 1994 i ro trcinio. Doctoral Thesis (Chemistry).
The absolute . : vat iabiliii and m ruveri idad Nationald i tcult
14 tctronlc acids Natural Ciencias- Bogota).
CTMJNIPOrre.oidGuui.nc.-tIDsILa.enStdtIeIr-s2.im\lNiini:Oea5r15-s.5c6&tcMrtIeNrpAenLeEsJfro1m97t2hae MARdIeIIcIaJNnIoEaMZtUe.IUAa,.bVraD1n9cU9h5QeadU,CcVah,raiianbCit,liaftilMyIAla-RcmiAedi.lensltNoecrtao&-f
marine 5ponge fa MCI OflOS, "Tetrahedron 2.S: furanosesterpene lelronic acid. From the sponge
197323b3.-3Fu>4r.ther linear ftira/TDterpenes from Marine MARTirIciNrI.i /, VN.aturOaUlQPrUoEdu,ctsCU,tteSrsAT6.O2,81-N2N4.X:
sponges, retrahedron 28:88
:dSe,feAn.sRe; Ie9v9i|.deAnlckealEocidslbandCCaoslaOdgiiaCnalchreolme "I" asIenIrdcrt{cIir.Ip.eine t-ctio"ni"ciac'idis firnlio3fm/i,:m0ar/rtkiesvnne-ofsbupiio1nigneisn
natural products from I oma oliVQcewn. h ma, t bcmical & Pharmaceutical
MarineBiologj III: 375-374. Bulletin 450 181484.
DAVISr.evRi.si&onCoAfPthOeNm,arRi.ne19s9e4s.leTrwteorpfeanreonLee:lrosnuiicllacid MARTINE&Z,UVI.MDOlL:TfoQ.UE, C. SATO, N1., TANAK.A.
-irobiliiiin to (8ZJ?E«20Z)-6tJttb!linin R. I > !8R)-variabiHn
(SL-,l3Z,20Z)-sirobiliuiri. Australian Journal of from the spong*e IrviruQ ff/iw Natural Products
Letters 6: It-.
DELCDheVetAmclir.smltirnIyn.c47i\:o!n.9r3d3cA436laV.AacRtGivAidSa,d uIn".alVjjes1i9ca97-v MURRaCAcAYiPd.ONLf.,roHmKA.MaI1n99I.5AHu.s,AtHraOnleOiwPaEnsRet,mearCrteiUrnpOecBncBSSpte,othLg.e&,
antiinflamaiorLt del i I )-eurcufenol y la ma inia y>. \ustraJwwi lournalofCTiemistn
Nviaai.re.iiaobnialiiliu;adeaisC'oaldoosmbdica,DShFepamocinusjltatrsa}dmMaldrei'nnaCisiv.eenrcs:iiadsad: r'\RR;I\S,: IP8,J99-]1999Q72..Apm.vimacion al posible papcl
tduccion de cnetabolhossecund-
Ota),
FAULsKp1oN2nE1g-eB3,8I2Ir2.c.i\n9i1avariaibiliistinT,eatnraahnteidbrioontiIcetttreormst3h9e^ aiDrLeionmsiovesenrpsoliandaegdsipNioaunejia>o,nmaaBlr.iSdn(eaC[orrlchoi$nmsnbiiifaifc,li(FxBai(cPouollrtiotagcdyr.)di,e.
AaB-c.OKtAiuvhuiiBdl.aMiSdnca,#aariTusPhli1emsuNii5csi5r0o(:bNPi,haa|nrIMamcasc>pelo<uniat<jn>ia7tc-iamtlCuaimrtCoiohrtnesaax!mii/c/'d.iedadl,a PAW!sF1 pEIoNKan.r1gieCas.As1:.L.a.gBa(oignwMost.Ata)Np,1rA9e9Sd5a,.torDIyeCf.reenctls"eOfsiOshNo.fLI.CNaC,rhiebmRbi.eia&nnl
it,
Unrveraidad Naeional de Colombia, Facuhadde detcrrencv Maj iogj Progress Series 127
( 183-194.
ncias Bogota).
GARSON, MJ, f994, Ihe biosynthesis of SJ* R{\I?LER, k. 1990. Associations beiueen Caribbean
427-i4&4d0a.qInmSciitaesbiolRr.tVaYM.i vb;an,rlKieimpimepno.rtTahn.t,MOpp.. 455-466. InmdRiitpehloort, K. (ed)i tNiceworpgearnsipcec^-t^n Pp
HI.J-l ngc btologj (Smithsonian Institution Press.
I
space. (Balkema: Rotterdam). ; Pan iIcm.
JAi iBl-Rl. J. I98J. Variations oftiie shape i SASINSllM.il INC 19 u I igtJfdl
chlorophyllconcentrationoftheseleractinianc^vaI release 6.03 edilion. (SAS Insiuule.
I
9ffV$rd Yernl: two COtllpI
696 MEMOIRS OF THE QUEENSLAND MUSEUM
SJIDDON, S, CHESHIRE K. & WILKINSON. C. (Demosnongiae: Poeciloselcrida). MarineBjolog)
1992 Photoplvysiology and acclimation of the 124: 583-590.
coral reefsponge Cymbasttstci concentricu across URIZ, M.. R/ROK X., GACERA, J. & FUR, J. VI..
a depth gradient. Pp. 847-852 In Proceeding 1996b. New light on the cell location of Avarol
the 7ih international Coral ReefSymposium. Vol. within the sponge Dysidea avara (Dendro-
SIMPS2.O1N1,niTv.eIr.s.itj198o4f.GuTahme:ceGlulambi)o,logy bfspoji VICENT;Ei,tidav),.pC.ell19&90.tisRseusepRoensseearcohf 2s8p5o:ngSIes9 will-.
(Springer-Verlag: New York). autotrophic endosymbionts during die coral-
SOKAL, R.R. & ROHLI-, E..L 1981. Biometry. 2nd bleachingepisode in Puerto Rich. Coral Reefs S.
Edition- AV.I I. Freeman: San Francisco). 199-202,
M'l.LIvan. IV. kaih KNER,DJ & W\ BBJ 1983, WALKER, R.P., THOMPSON, J.L. & FAULKNBR,
Siphonodtctine, a metabolite ol the burrowing D..L L9&5. Exudation oi illy-active
sponge Siphonodivtyon sp. thai inhibits coral metabolites in the sponge \pi\siua fistularfcs II.
iwth. Science221; 1175-1176. Chemical evidence. Marine Biology 88; 27-32.
IIIOMPSON, J.r. 1985. Exudation of biologically- WILKINSON. C.R. 1980. Cyanobactena symbiotic in
-aetive metabolite*; in the sponge ipfysma ine sponges. Pp. 553-563. InSchweninger \V.
fistulctris L Biological evidence. Marine Bi«i & Schenk, H.E.A teds) Endoeytobiology. endo-
rHOM8P8:S2O3N-2,6,i-i -. BARROW, K..D.& FAULKNER, Cruvlbeiros:isBeralnidn).eel! biology. Vol. 1 (Walter de
mDe.tJa.bol1i9t8e5s., aLeorcoatlhiizonaitnioanndohfomtowaoerobtrhoimoinnian,teidn WILKINSON, C.R.. GARRONE, R. & \AC! LI- C I
984. Marine spongesdiscriminate between lood
us cells ofthe marine sponge Ipfysiria b1acteria and bacterial symbionts: electronmicro-
(arts {~l't'rongit! thianu). Acta /oolouica cope radioautograph) and \n s/fw evidence.
11IOM&(vaSPrtESioaVOctAkNiohN,noSlM,im.n)FEd..6i,4At(.Me4rL)p1I:e0Rn81eP79Hc9Yo-I2m,n1pv0oPi.sLri,otnimBoeEnnRtoQeftlJlthvteSiTnmd,airPi.nRe. WILK2PI2r0oN(c1eS2eO2d1iN)n,:gs51oC9f.-R5t.h2e8.&RovaYlA<SocLiIetLyIo,t" LJ.ond|o9n79B
sponge Rhnpalo >,ihile. Biochemical Transplantation of marine sponges to different
conditions of light and current. Journal of
Systematica and cology 15: 595-606.
IIIOMPSON, .Li .. WAI LKER, R.R & FAULKNER, 4E1x-p1e0r4i.mental Marine Biology and Ecology 37:
TV.!. 1985.. Screening and bioassays for
biologtcally-active substances from forty marine WITTAKER. R.H.& FEENY,P.P. 1971.Allelochemics-
sponges h ies from San Diego, California, uiic.il interactions between species. Science
USA. MarineBiology88: II 171:757-770.
miYANov. i: I9S1. Adaptation of reefbuilding WULFF, J. 1904. Sponge feeding by Caribbean
corals to low light intensity. Pp. 39-43. In angeifishcs. trankfishes, and ftlefishes. Pp. 256-
SPryomcpeoesdiiunmgs. oVfolT.he2.4t(hi Iinternationoafl PChoirlailppRieneefs.: &' :BrIaneSkomeaaul,R..fW-.(_M..(veadns,) KSepmopnegne.s Tihn.tMimGe. vMaidn
Manila space. (Balkema: Rotterdam).
I.
II 'RON,X..BECERRO, M.& 11R1Z, M- 199< 199 loi . (ceding by the Caribbean starfish
patterns of toxicity in benthlc invertebrates: the Maiine BlOlog) 1 23.
encrusting sponge ( ^rambc erQWlbt s(3- ,
1 1RBA(NPo,ecSi.lo&scClcArPidOaN)..ORl.ko1992.Comi e40l.ms(ACI new ZEA,ifS.ata1l9o8g7O.CiEesntpiolnijcan; Bolegi ia]ribe Coiombiano:
loscsterterpenes from an Australian marine 1994. Patterns of coral and sponge abundance in
sponge, Spongia sp. Australian Journal ol' stressed coral reef's at Santfl Maria Colombian
Chemistrv 45: 1255-1263. Caribbean. Pp. 257-264. In Soesi R.W'.M. van,
OKI/.M,LHE< LRRO.M.A., H IK..J.M.& it RON. Kempen, Th.M.G. van & Braekman, J.-C. (eds)
X. 1996a. Location of toxicity within "he Sponges in ti-nn ind space (Balkema:
Mcditer' tsa \mbn crumb* Rotterdam}.
