Chemical control of epibiosis by Hong Kong sponges: the effect of sponge extracts on micro- and macrofouling communities

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1 MARINE ECOLOGY PROGRESS SERIES Vol. 297: , 2005 Pulished August 1 Mr Eol Prog Ser Chemil ontrol of epiiosis y Hong Kong sponges: the effet of sponge extrts on miro- nd mrofouling ommunities Sergey Doretsov, Hns-Uwe Dhms, Mndy Y. Tsoi, Pei-Yun Qin* Mrine Costl Lortory nd Deprtment of Biology, Hong Kong University of Siene nd Tehnology, Cler Wter By, Kowloon, Hong Kong SAR ABSTRACT: The reltionship etween ntifouling metolite prodution nd epiiosis on the surfes of the sponges Hlilon ymeformis, Hlilon sp. nd Cllyspongi sp. ws investigted in this study. Densities of mrofoulers nd ditoms were suppressed on the surfes of ll exmined sponges, while densities of teri on the surfes of H. ymeformis nd Cllyspongi sp. were similr to those on the referene surfes nd were more thn doule the densities of teri on the surfes of Hlilon sp. ompred with referene surfe. Bry-Curtis similrity mtries of the trflp (terminl restrition frgment length polymorphism) nlysis of PCR-mplified teril 16S rrna genes otined from the surfes of the sponges demonstrted tht the teril ommunities on the sponge surfes were different from eh other nd from those on the referene surfes. In field experiments, oth methnol nd dihloromethne extrts from ll tested sponges inorported in Phytgel mtrix inhiited reruitment of ditoms, lge nd invertertes, ut extrts of only 2 sponges deterred teril film development. The trflp nlysis reveled tht the sponge extrts deresed diversity in the teril ommunity. Strong negtive effets of the sponge extrts on the Shnnon-Wiener diversity vlues, s well s the speies-rihness vlues of the ditom ommunity were found. ANOSIM (nlysis of similrity) nd SIMPER (similrity perentge) nlyses demonstrted tht the type nd speies speifiity of the extrts ffeted the ditom omposition. Results suggest tht metolites of sponges n ontrol reruitment of propgules, hnge the omposition of miro- nd mrofouling ommunities nd, in this wy, regulte epiiosis on sponge surfes. KEY WORDS: Sponge Chemil defense Seondry metolites Mirofouling ommunity Mrofouling ommunity Bteri Ditoms Epiiosis Resle or repulition not permitted without written onsent of the pulisher INTRODUCTION Surfes in the mrine environment re ontinuously exposed to nd olonized y miroorgnisms nd propgules of metellulr orgnisms (Whl 1989). As result, omplex ommunities of fouling orgnisms populte mny mrine surfes. Bteri nd ditoms re mong the first orgnisms tht tth onto surfes (Chrklis & Cooksey 1983) nd form nturl iofilms tht re highly vrile over time nd heterogeneous in omposition (Qin et l. 2003). The settlement of orgnisms on the surfes of living orgnisms (i.e. epiiosis) n e oth dvntgeous nd disdvntgeous to the host. Advntges ould e the prodution of ntifouling sustnes y symioti epiiot (Wlls et l. 1993, Hrder et l. 2003, Piel 2004) nd furnishing the host with nutrients (Merdo et l. 1998, Fulkner et l. 2000). The disdvntges inlude the possile inhiition of growth, nerosis, or deth of host orgnisms (Whl & Mrk 1999). On soft-odied mrine orgnisms, suh s sponges, the extent of miroil oloniztion is possily influened y the hemil effets of iotive metolites produed either y the host itself or y symioti miroorgnisms (Lee et l. *Corresponding uthor. Emil: oqinpy@ust.hk Inter-Reserh

2 120 Mr Eol Prog Ser 297: , , Kelly et l. 2003). Suh hemil effets led to the formtion of the distintive miroil ommunities tht re ssoited with sponges. Severl investigtions hve shown tht the miroiot from sponges differ from those ssoited with non-living sustrt in the mient proximity (Hentshel et l. 2003, Thoms et l. 2003, Lee & Qin 2004, Tylor et l. 2004), suggesting tht miroilsponge ssoitions re speifi. Hentshel et l. (2002) demonstrted, for exmple, tht the sponges Aplysin eropho nd Theonell swinhoei hve uniform internl miroil ommunity tht is different from the miroil ommunity in the mient environment of the sponges nd from miroil plnkton. Dt ville on the density of epiioti teri on the surfe of sponges re limited nd inomplete. Bteril numers hve een estimted t 6.4 ± g 1 tissue of the sponge Aplysin eropho (Friedrih et l. 2001) nd to ml 1 sponge extrt for Rhoploides odorle (Wester & Hill 2001). It hs een shown tht the numer of olony-forming teri from the surfe of the sponge Irini rmos rnged from 7 to olony-forming units m 2 (Thkur & Anil 2000). Sponges re known to e rih soures of unique nd diverse iotive metolites tht provide potent ntiteril, ntifungl, ntifeeding, nd/or ntifouling protetion (Austin 2001, Ser et l. 1999, Fulkner 2000, Blunt et l. 2003). At the sme time, there is only limited evidene s yet on the funtionlity of these ompounds in the nturl environment ginst nturlly ourring ntgonists, suh s miro- nd mrofoulers. In previous lortory nd field experiments, we demonstrted tht extrts from the 7 dominnt sponge speies in Hong Kong wters hve the potentil to redue or inhiit the reruitment of teri nd ditoms in the se (Doretsov et l. 2005). The sponge Cllyspongi pulvint n ffet the reruitment of miro- nd mrofoulers not only on its surfe, ut lso on non-living sustrtes nery (Doretsov et l. 2004). The sponge speies Hlilon ymeformis, Hlilon sp. nd Cllyspongi sp. re rrely ffeted y fouling in Hong Kong ostl wters. From preliminry oservtions, we hypothesized tht these sponges nd/or their miroil ssoites produe ompounds tht n ffet the oloniztion of miro- nd mroorgnisms y inresing the undne of some speies nd deresing the undne of others. In the present study, we qulittively nd quntittively ompred teril ommunities on sponge surfes nd on innimte surfes. For the first time, snning eletron mirosopy (SEM) ws used to visulize teri on the surfes of the sponges. Crude extrts of the sponges were inorported in Phytgel mtrix (f. Henrikson & Pwlik 1995) tht ws susequently deployed s sustrtum for tthment nd oloniztion y teri, ditoms, mrolge nd invertertes under field onditions. The teril ommunity profiles developed on mtrix surfes t the end of the experiments were ompred y the ulture-independent, PCR-sed moleulr fingerprinting tehnique of terminl restrition frgment length polymorphism (trflp) (Liu et l. 1997). This pproh llowed us to overome the disdvntges ssoited with ulturedependent tehniques nd reveled the effets of sponge metolites on teril oloniztion under nturl onditions (Hrder et l. 2004). In the present study, we posed the following questions: (1) Are there differenes in the density nd omposition of miroil ommunities on the surfes of the 3 sponge speies Hlilon ymeformis, Hlilon sp. nd Cllyspongi sp. ompred to innimte surfes? (2) Do sponge extrts ffet the formtion of mirofouling nd mrofouling ommunities in field experiments? (3) Is there orrespondene etween epiiosis on the surfes of sponges nd the ntifouling tivity of sponge extrts? MATERIALS AND METHODS Colletion of sponges nd preprtion of extrts. Speimens of the sponges Hlilon ymeformis (ZMAPOR 17602), Hlilon sp. (ZMAPOR 17600) nd Cllyspongi sp. (ZMAPOR 17596) were olleted from Hong Kong ostl wters t depths of 1 to 3 m, ner the Hong Kong University of Siene nd Tehnology Pier (22 21 N, E). The sponges were refully retrieved to the wter surfe. Five sponge speies speimens were ut into severl portions. One piee of out 10 ml iovolume of eh sponge ws used for trflp nlysis. Another portion ws stored in 70% ethnol for susequent txonomi identifition y Prof. Ro vn Soest (Zoologil Museum, University of Amsterdm). A smll intt piee of sponge tissue ws fixed in 3% glutrldehyde (in rtifiil sewter free of lium nd mgnesium) for SEM nlysis. We mesured the wet weight of the mjor prt of the sponge (>500 g) t the pier, nd the tissue volume ws mesured y wter displement. The sponge speies were trnsferred to lrge ukets with erted sewter nd rought to the lortory. In the lortory, the remining intt sponge tissues were ut into 2 equl portions of wet weight of out 180 to 200 g. One portion ws extrted twie with dihloromethne (DCM, Fisher Chemils) for 8 h with gentle gittion; the other portion ws extrted with 99.9% methnol (MeOH, Merk) in the sme wy. The extrts were filtered through Whtmn No. 1 filter

3 Doretsov et l.: Antifouling tivity of sponges 121 pper nd redued y rotry evportion to onentrtion tht ws volumetrilly equivlent to 10% of the originl smple tissue. Tissue-level onentrtions (Jensen et l. 1996) were djusted prior to the experiments desried elow. Gel-immoiliztion of sponge tissue extrts. The method for immoiliztion of the orgni tissue extrts into gels ws dpted from tht desried y Henrikson & Pwlik (1998) nd Hrder et l. (2004), with slight modifition. The sponge extrts were dissolved in dimethyl sulfoxide (DMSO) t onentrtion 10 times tht of the tissue level. Gels were prepred y dding Phytgel (Sigm Chemil) to stirred eker ontining oiling doule-distilled wter to yield 4% (w/v) gel onentrtion. After the gel solution ooled down to 70 C, the rude extrts were diluted to tissue-level onentrtions with the gel solution nd vigorously mixed for even distriution of the extrt in the gel. A ontrol ws prepred with pure DMSO solution insted of the rude extrts. Finlly, 5-ml portions of the hot Phytgel solution were poured to Flon Petri dishes nd llowed to solidify slowly. The Petri dishes were stored overnight t 4 C to llow for homogeneous diffusion of the extrt omponents throughout the gel. These dishes were used in the experiments desried elow. Snning eletron mirosopy. Smll piees of the 3 sponge speies nd referene sustrt (stones nd shells from the lose viinity of the sponges) were dehydrted in n lohol series, dried y ritil-point drying nd oted with gold (for detils see Doretsov & Qin 2002). The speimens were then exmined y JEOL 6300F (70 ev) snning eletron mirosope. Bteril ounts were mde y 6 rndomly seleted fields of view (squre = 500 µm 2 ) per replite. The densities of the teri were lulted s the numer of ells per squre millimeter of the sponge surfe. Field experiments. Gel-oted Petri dishes were exposed to fouling during 3 wk t the Hong Kong University Siene nd Tehnology Pier (22 21 N, E) t depth 1 m elow the low wter mrk. The long exposure time in our experiment ws neessitted y the low fouling rte during the winter period in Hong Kong wters (Qiu et l. 2003). There were 13 replites per tretment. After the first week, 8 Petri dishes were hrvested. Five of them were then fixed in 4% formldehyde solution in sewter for the susequent enumertion of teri nd ditoms. The dominnt speies of ditoms on the dishes were determined in 10 rndomly seleted fields of view under the mirosope (Axiophot, Zeiss; mgnifition: 400 ) using key to the mrine enthi ditoms of Chin (Jin et l. 1985). The tthed teri were visulized y the DNA-inding fluorohrome 4, 6-dimidino-2- phenylindole (DAPI, Fluk Chemie) t 0.5 µg ml 1. Formlin-fixed (4% in filtered [0.22 µm] sewter [FSW]) dishes were rinsed with utolved (120 C for 30 min) FSW nd stined with DAPI for 15 min. The numer of teri in 5 rndomly seleted fields of view ws estimted y epifluoresene mirosopy (Olympus, Jpn, mgnifition: 1000 ; λ Ex = 359 nm, λ Em = 441 nm). Three other Petri dishes were used for the nlysis of teril ommunities developed on the dishes. After 3 wk, 5 dditionl Petri dishes were fixed in 4% formldehyde solution in sewter for susequent nlysis of the mrofouling ommunities. Densities of the settlers (ind. m 2 ) were determined under the mirosope (mgnifition: 0.63 ). Anlysis of teril ommunities. We ompred teril ommunities y trflp nlysis (Liu et l. 1997). For this purpose, the entire surfe re ( m 2 ) of the experimentl nd ontrol Petri dishes or 10.0 m 2 surfe res of the sponges nd referene sustrt were ompletely swed with sterile otton uds. Sws from eh gel were individully suspended in 1 ml of extrtion uffer (100 mm Tris- HCl, 100 mm EDTA, 100 mm sodium phosphte, 1.5 M sodium hloride, 1% CTAB; t ph 8) in 2-ml miroentrifuge tues. For lysing, the smples were sujeted to 3 yles of freezing nd thwing followed y 2 h of inution in 20% sodium dodeylsulfte (SDS) t 65 C. The otton uds were removed, nd, fter entrifugtion ( rpm for 5 min), the totl DNA in the superntnt ws extrted nd purified twie in volume of 24:1 hloroform:isomyl-lohol, followed y preipittion in isopropnol t room temperture for 15 min. The preipitted DNA ws wshed with old 70% ethnol nd resuspended in 50 µl of utolved, doule-distilled wter nd frozen until use. The 16S rrna genes (rdna) of the teril ommunity were mplified y polymerse hin retion (PCR) in totl volume of 25 µl, ontining 1 µl of DNA templte, 250 µm of eh desoxyrionuleotide triphosphte (datp, dctp, dgtp, dttp; Phrmi Biotehnology, USA), 1 U of DNA Tq polymerse (Amershm Biosienes, USA) nd 0.8 µm of eh universl primer: 341F forwrd (5 -CCTACGGGAGGCA- GCAG-3 ) nd 926R reversl (5 -CCGTCAATTCCTT- TRAGTTT-3 ) (Amnn et l. 1990, Lee et l. 1993). The 926R primer ws leled t the 5 -end with 6-roxy fluoresein (FAM) dye. The thermoyling onditions were s follows: hot strt t 95 C for 2 min (1 yle) nd 15 yles of 95 C for 30 s, 56 C for 3 min nd 72 C for 3 min. The nneling temperture strted t 56 C nd ws redued to 40 C in inrements of 1 C yle 1. The mplified DNA (4 µl of the PCR mixtures) ws visulized y gel eletrophoresis on 1.5% grose gel in Tris-etteethylenediminetetreti id (TAE) uffer.

4 122 Mr Eol Prog Ser 297: , 2005 Fluoresently leled PCR produts were then purified with the Wizrd PCR preps DNA purifition system (Promeg) ording to the mnufturer s protool. Purified mplions were digested with 20 U MspI (Boehringer Mnnheim Biohemils) t 37 C for 6 h. Aliquots of digested produts (10 µl) were mixed with 0.5 µl of internl size stndrd (ET550-R, Amershm Biosienes). This mixture ws dentured for 2 min t 95 C nd immeditely hilled on ie prior to pillry eletrophoresis on MegBACE geneti nlyzer (Amershm Biosienes) operted in the genotyping mode. After eletrophoresis, the lengths of the fluoresently leled terminl restrition frgments (TRFs) were determined y omprison with internl size stndrds y using the softwre Frgment Profiler (Amershm Biosienes). TRFs tht differed y <1 p were onsidered identil. Frgment lengths tht were present in 3 smples were used to produe representtive TRF profile of eh teril ommunity. Sttistil nlyses. The densities of teri, ditoms nd mrofoulers on the Petri dishes nd on the surfes of the sponges were log-trnsformed in order to ensure normlity of vrine (Zr 1999). In the se of the sene of ditoms in field of view, vlue of log(y+1) ws ssigned to improve the trnsformtion. In ll ses, the normlity ssumption ws verified with the Shpiro-Wilk test (Shpiro & Wilk 1965). The differenes etween the experimentl nd ontrol tretments were determined y 1-wy ANOVA, followed y the Tukey HSD (honestly signifint differenes) post ho test (Zr 1999). In ll ses, the threshold for signifine ws 5%. TRF ptterns of different teril ommunity DNA smples were sujeted to luster nlysis. Bry-Curtis similrities were used to produe similrity mtrix sed on the totl numer of TRFs oserved in ll smples nd the presene or sene of these TRFs in individul smples. For the onstrution of dendrogrm demrting the similrity of miroil ommunities on the gels, group verge linkge in the hierrhil, gglomertive lustering lgorithm ws performed using the PRIMER progrm (Plymouth Mrine Lortory, UK). As mesure of ditom nd mrofouling speies diversity, Shnnon s diversity index (H ) nd evenness (Wrwik & Clrke 1995, Clrke & Gorley 2001) were lulted y using PRIMER. Mrglef s speies rihness (Wrwik & Clrke 1995) ws lso lulted. Similrity perentge nlysis (SIMPER) (Clrke 1993) ws used to determine the tx ontriuting most to the dissimilrity etween groups. All these nlyses were done using the Bry-Curtis similrity oeffiient (Clrke 1993) pplied to the 4th-root-trnsformed ditom speies-undne dt. Anlyses were performed using the PRIMER softwre pkge. Assemlge similrity ws ompred etween the sponge speies (sponge effet) nd mong the different solvents (solvent effet) using 2-wy ANOSIM (Clrke & Wrwik 1994). RESULTS Miroil epiiosis on the surfes of sponges The densities of epiioti teri reveled y SEM on the surfes of Hlilon sp. were higher (ANOVA, F = 17.8; HSD, p < 0.05) thn on the surfes of other sponges nd on the referene surfes (Fig. 1). The densities of epiioti teri on the surfes of H. ymeformis nd Cllyspongi sp. were not signifintly different from eh other or from the referene surfes (ANOVA; HSD, p > 0.05). No invertertes, ditoms, or mrolge were found to e tthed onto ny surfe of the sponges, while ditoms (density: 2000 ells mm 2 ), the tueworm Hydroides elegns (density: 3.2 ind. m 2 ) nd the rnle Blnus trigonus (density: 0.2 ind. m 2 ) were dominnt on the referene surfes. Bteril ommunity profiles on sponge surfes Terminl restrition frgment length polymorphism reveled tht the teril ommunity profiles were different on the sponge surfes nd on the innimte (referene) surfes. The lowest numer of teril Bteril densities x10 3 ells mm Hlilon sp. Cllyspongi sp. H. ymeformis Referene Fig. 1. Bteril densities (ells mm 2 ) on the surfes of the sponges Hlilon sp., Cllyspongi sp. nd Hlilon ymeformis nd referene site (stones nd mussel shells from the lose viinity >0.5 m). Brs indite men ± SE of 5 replites. Dt tht re signifintly different ording to Tukey test (ANOVA: p < 0.05) re indited y different letters ove the rs

5 Doretsov et l.: Antifouling tivity of sponges 123 riotypes (TRFs = 28) ws reorded from the referene surfes, while the teril ommunities tht developed on the surfes of the sponges Hlilon sp. nd H. ymeformis hd doule the numer of teril riotypes. The highest numer of riotypes (TRFs = 73) ws found in the teril ommunities from the surfes of Cllyspongi sp. Bteril ommunities tht developed on the surfes of the sponges hd speifi TRFs tht were not found on referene surfes (e.g. 43, 122 nd 247 p). At the sme time, eh sponge hd speifi TRFs tht ould not e found on the surfes of the other sponges: e.g. H. ymeformis hd speifi TRF t 138 p; Cllyspongi sp., t 314 p; nd Hlilon sp., t 72 p. Hlilon sp. nd H. ymeformis shred similr TRFs t 120 nd 313 p. The presene of ommon TRFs t 245, 312, 367, 369 nd 420 p on ll sponge nd referene surfes might indite tht some teril speies were not ffeted y sponge metolites. Aording to the Bry-Curtis similrity mtries, sed on the presene (indited y 1) or sene (indited y 0) of given TRF in pttern, the teril ommunities were divided into severl groups (Fig. 2). The teril ommunities tht developed on the referene surfes hd low similrity (<40%) ompred with the teril ommunities tht developed on the sponge surfes, whih formed seprte lusters. Bteril ommunities from the surfe of Hlilon sp. nd H. ymeformis shred some similrities nd ould e omined into 1 su-luster in the dendrogrm, while ommunities from the surfe of Cllyspongi sp. were different from ommunities on the surfes of the other sponges. Similrity % Referene 1 Referene 2 Referene 3 Cllyspongi sp. 1 Cllyspongi sp. 2 Cllyspongi sp. 3 Fig. 2. Cluster nlysis inditing similrities etween teril ommunities found on the surfe of the sponges Hlilon sp., Cllyspongi sp. nd H. ymeformis nd referene site (stones from the lose viinity). Different replites (n = 3) identified s orresponding numers H.ymeformis 1 H.ymeformis 2 H.ymeformis 3 Hlilon sp. 1 Hlilon sp. 2 Hlilon sp. 3 Effet of sponge metolites on teril ommunities developed on gels After 7 d of the experiments, rod-shped nd ooid teri dominted the teril ommunities on the gels. Epifluoresene mirosopil ounting reveled tht the densities of teri on the gel surfes were signifintly different mong the tretments (Fig. 3A: ANOVA, F = 14.7, p < 0.05). The highest density of teri ws found on the ontrol dishes nd on dishes with MeOH nd DCM extrts of the sponge Hlilon ymeformis. The lowest densities of teri were disernile on dishes with MeOH extrts of the sponge Hlilon sp., wheres dishes with MeOH nd DCM extrts of Cllyspongi sp. hd moderte densities of teri (Fig. 3A). The MeOH nd DCM extrts Bteril densities x 10 3 ells x mm 2 Ditom densities x 10 2 ells mm A B d Hlilon sp. D Cllyspongi sp. MeOH DCM H. ymeformis Control Fig. 3. Densities of (A) teri nd (B) ditoms tht developed on the surfe of Phytgel gels fter 7 d of exposure t the university pier t depth of 1 m elow the low wter mrk. The gels were enrihed with immoilized methnol (MeOH) nd dihloromethne (DCM) extrts from the sponges Hlilon sp., H. ymeformis nd Cllyspongi sp. In the ontrol, 4% (v/v) solution of DMSO ws used. Brs indite men ± SE of 5 replites. Dt tht re signifintly different ording to Tukey test (ANOVA: p < 0.05) re indited y different letters ove the rs

6 124 Mr Eol Prog Ser 297: , 2005 Fig. 4. Cluster nlysis of the similrity etween teril ommunities tht developed on the surfes of Phytgel gels with immoilized sponge extrts of Hlilon sp. (H), H. ymeformis (H) nd Cllyspongi sp. (C) derived from methnol (Me) nd dihloromethne (DCM) extrtion fter 7 d of exposure t the university pier t depth of 1 m elow the low wter mrk. Different replites (n = 3) identified y orresponding numers. In the ontrol (Con), 4% (v/v) solution of DMSO ws used of the sponges eqully ffeted teril tthment to the dishes. The mximl numer of teril riotypes (TRFs = 166) ws found on the ontrol gels, wheres the miniml numer (TRFs = 35) ws oserved on gels with MeOH extrts of Cllyspongi sp. Other gels hd moderte numer of TRFs. Asene s well s presene of ertin TRFs hrterized the teril ommunities developed on dishes with or without sponge extrts. TRFs with lengths of 129, 235, 236 nd 240 p were found only on the ontrol gels. Some teri were identified tht were not ffeted y sponge metolites; their riotypes (prtiulrly 120, 312 nd 584 p) were present on oth the ontrol nd on the experimentl gels. Aording to the Bry-Curtis similrity mtries, the teril ommunities ould e divided into severl groups (Fig. 4). The teril ommunities developed on the ontrol gels with DCM nd MeOH extrts hd low similrity (out 30%) ompred with the teril ommunities formed on gels with MeOH nd DCM extrts of the sponges. We found similr teril ommunities on gels with extrts of Hlilon ymeformis nd Hlilon sp. Bteril ommunities on the gels with MeOH nd DCM extrts of Cllyspongi sp. were different from eh other nd from the teril ommunities developed on the gels with extrts of H. ymeformis nd Hlilon sp. Effet of sponge metolites on ditom ommunities After 7 d, 12 speies of ditoms were found on the gels, inluding Nitzshi longissim, N. onstrit, Nitzshi sp. 1, Billri sp., Limophor sp., Nviul sp. 1, Nviul sp. 2, Nviul sp. 3, Pleurostigm sp., Diploneis sp., Mstogloi pumil nd Ahnntes sp. We oserved the highest densities of ditoms on the ontrol gels. The densities of ditoms on the gels with sponge extrts were signifintly lower thn those on the ontrol gels (Fig. 3B: ANOVA, F = 9.3; HSD, p < 0.05). On the surfe of gels with MeOH extrts of Hlilon sp., we oserved only 1 speies, the ditom N. longissim, t very low densities. The densities of ditoms on the gels with MeOH extrts of Cllyspongi sp. nd H. ymeformis were moderte. The MeOH nd DCM extrts of the sponges ffeted the density of the ditoms in similr wy, with the exeption of extrts of Cllyspongi sp. The ditom ommunities tht developed on the surfes of the ontrol gels with DCM nd MeOH extrts were similr nd were grouped in seprte lusters ording to the Bry-Curtis similrity mtrix (Fig. 5). The ditom ommunities tht formed on the gels with MeOH nd DCM extrts of the sponges were different from eh other nd formed seprte groups. The ditom ommunities from gels with the MeOH extrt of Hlilon sp. hd low similrity (20%) with the other ditom ommunities, forming n independent luster, while the DCM extrts of this sponge were similr to the DCM extrts of H. ymeformis. Com- Fig. 5. Cluster nlysis of the similrity etween ditom ommunities tht developed on the surfes of Phytgel gels with immoilized sponge extrts of Hlilon sp. (H), H. ymeformis (H) nd Cllyspongi sp. (C) derived from methnol (Me) nd dihloromethne (DCM) extrtion fter 7 d of exposure t the university pier t depth of 1 m elow the low wter mrk. Different replites (n = 5) re identified y orresponding numers. In the ontrol (Con), 4% (v/v) solution of DMSO ws used

7 Doretsov et l.: Antifouling tivity of sponges 125 munities developed in the presene of DCM nd MeOH extrts of Cllyspongi sp. formed distintly relted lusters in the dendrogrm. The ANOSIM test provided evidene tht the ditom ommunities on the gels were signifintly different ording to sponge speies (tretment effet: glol R = 0.72, p = 0.001) nd the method of extrtion (extrt effet: glol R = 0.35, p = 0.04) (Tle 1). The SIMPER nlysis reveled tht 6 ditom speies ounted for >84% of the totl Bry-Curtis dissimilrity mong ommunities from different tretments. The ditom speies Nitzshi longissim nd Nviul sp. 1 ounted for lmost 50% of the totl etween-group dissimilrity. The highest rihness nd diversity in the ditom ommunities were found on the ontrol dishes nd dishes with DCM extrts of Hlilon sp. nd Hlihondri sp. (Fig. 6A,B: ANOVA, F = 60.95; HSD, p < 0.01 nd ANOVA, F = 72.02; HSD, p < 0.01, respetively). The lowest diversity nd rihness were found on dishes with MeOH extrts of Hlilon sp. The lowest evenness ws oserved in the ditom ommunities formed in the presene of MeOH extrts of Hlilon sp., while the evenness of other ommunities formed in the presene of sponge extrts ws similr. Effet of sponge metolites on mrofouling ommunities Tle 1. Results of ANOSIM (glol R, p) nd SIMPER nlysis on the ditom speies omposition on dishes with Phytgel ontining methnol or dihloromethne extrts (extrt effet) of sponges nd ontrol dishes (tretment effet). The perentge ontriution of eh ftor is verged over ll signifint omprisons Speies Tretment effet (%) Extrt effet (%) Glol R = 0.72, Glol R = 0.35, p = p = 0.04 Nitzshi longissim Nviul sp Nviul sp Billri sp Diploneis sp Ahnnthes sp Other speies After 3 wk of experiments, we found 1 rown lg (Pheophyt: Etorples, unidentified rown lg) nd 5 inverterte speies (the hydrozon Oeli sp. [Cnidri: Hydrozo]; 2 polyhete speies Hydroides elegns nd Spiroris sp. [Annelid: Polyhet]; the rnle speies Blnus mphitrite [Cruste: Cirripedi]; nd the ryozon speies Bugul neritin [Bryozo: Cheilostomt]) on the experimentl pltes. The dominnt speies on the dishes were the tueworm H. elegns nd the rnle B. mphitrite (Fig. 7). Overll, the settlement of mrofoulers ws low, whih ws not surprising in the old seson in Hong Kong Rihness Diversity H Evenness A B C Hlilon sp. Cllyspongi sp. H.ymeformis MeOH DCM Control Fig. 6. Effet of methnol (MeOH) nd dihloromethne (DCM) extrts of the sponges Hlilon sp., H. ymeformis nd Cllyspongi sp. on (A) rihness, (B) diversity nd (C) speies evenness of ditom ommunities fter 7 d of exposure t the university pier t depth of 1 m elow the low wter mrk. Dt re expressed s mens ± SE of 5 replites. Dt tht re signifintly different ording to Tukey test (ANOVA: p < 0.05) re indited y different letters ove the rs

8 126 Mr Eol Prog Ser 297: , 2005 Men numer of settlers ( 10 2 ind m -2 ) Hydroides elegns Bugul neritin Blnus mphitrite Oeli sp. Spiroris sp. Brown lge ontrol-meoh ontrol-dcm Hlilon-MeOH Hlilon-DCM H.ymeformis-MeOH H.ymeformis-DCM wters (Qiu et l. 2003). Nevertheless, we were le to oserve the differenes in the settlement of mrofoulers on gels with nd without sponge extrts (ANOVA, F = 35.67, p < 0.05). The highest densities of settlers (HSD, p < 0.05) were found on the ontrol dishes, while the lowest settlement density ws oserved on dishes with sponge extrts. There ws no signifint differene in the fouling of the gels with MeOH nd DCM extrts of the different sponges. DISCUSSION Cllyspongi-MeOH Cllyspongi-DCM Fig. 7. Densities of mrofoulers (ind. m 2 ) on the surfe of gels with methnol (MeOH) nd dihloromethne (DCM) extrts of the sponges Hlilon sp., H. ymeformis nd Cllyspongi sp. or without them (ontrol) fter 3 wk of exposure t the university pier t depth of 1 m elow the low wter mrk. Dt re expressed s mens ± SE of 5 replites. Dt tht re signifintly different ording to Tukey test (ANOVA: p < 0.05) re indited y different letters ove the rs The present investigtion foused on the reltionship etween the ntifouling tivity of Hlilon ymeformis, Hlilon sp. nd Cllyspongi sp. extrts nd epiiosis on the surfes of these sponges. Our investigtion demonstrted tht the teril diversity on the surfes of the sponges ws higher thn the teril diversity on innimte surfes nd tht the teril omposition on the surfes of the sponges nd n innimte surfe ws different s well. The density of epiioti teri, ording to the SEM pitures, ws similr, exept on the sponge Hlilon sp., whih hd doule the density of teri. We did not detet ny ditoms or mrofouling invertertes on the surfes of the sponges. These results suggest tht sponges n regulte the omposition of miroil ommunities nd the ssemlges of mroorgnisms on their surfes nd tht some teri my e speifilly ssoited with prtiulr speies of sponges. Similr results hve een oserved y other investigtors. Chloroflexi teri hve een found to e speifi to ll Aplysin speies (Hentshel et l. 2003). The sponges Aplysin eropho nd Theonell swinhoei from the Mediterrnen Se, Red Se nd Pifi Oen hve uniform miroil ommunity tht is phylogenetilly different from tht of mrine plnkton nd mrine sediments (Hentshel et l. 2002). Moreover, internl (Burj & Hill 2001, Tylor et l. 2004) nd externl (Lee & Qin 2003, 2004) teril ommunities ssoited with sponges re different from teril ommunities on innimte ojets in lose viinity to the sponges. Some sponges hve een shown to ontin lrge numers of teri (up to 57% of the sponge volume, f. Hentshel et l. 2003). This ft my explin the high density of epiioti teri (out ells mm 2 ) tht we found on the surfe of Hlilon sp. ompred with the teri tht we oserved on the innimte surfes nd on the surfes of the other sponge speies. No previous work hs een done on the density of epiioti teri nd other miroorgnisms on the surfes of other sponge speies, with 3 exeptions. Kelmn et l. (2001) reported tht no teri were found on the surfe of Amphimedon viridis. Thkur & Anil (2000) onluded tht the numer of olony-forming teri on the surfe of the sponge Irini rmos rnged from 7 to olonyforming units m 2. Sine most of teri do not redily form olonies on stndrd gr medi (Hentshel et l. 2003), Thkur & Anil s (2000) experimentl work my provide only rough estimte of tul teril undnes on the surfes of sponges. Finlly, Amsler et l. (2000) found low density of ditoms ws on the surfes of the Arti sponges Dendrill memrnos, Leuett leptorhpsis nd Homxinell lfourensis in Jnury. Although our investigtion provides initil evidene on the densities of epiioti miroorgnisms on the surfes of some sponges, further investigtions re neessry in order to ompre densities of epiionts on different speies of sponges nd nery sustrt. The density of the teri on the surfes of the gels with the extrts of the sponges Hlilon sp. nd Cllyspongi sp. ws lower thn tht on the surfes of the ontrol gels nd on gels with the extrts of H. ymeformis. The teril ommunities tht developed on the ontrol nd experimentl gels were different from eh other nd were hrterized y the sene of ertin terminl restrition frgments nd the presene of others. Overll, the numer of riotypes on the surfe of the experimentl gels ws

9 Doretsov et l.: Antifouling tivity of sponges 127 lower thn tht on the surfe of the ontrol gel. Similr results were otined in our previous experiments with rude extrts of sponges from Hong Kong nd Hinn (Hrder et l. 2004, Doretsov et l. 2005). These differenes my e used y the ntiteril tivity of the sponge or y sponge-symiont-derived metolites, whih derese the density nd the diversity of the teri. In our experiments, teril ommunity profiles were ompred y using ulture-independent pproh, trflp. This fingerprinting method is reltively fst nd esy wy to provide semi-quntittive snpshot of ommunity diversity (Fuhrmn et l. 2002). It permits quik omprisons of different ommunities, providing minimum estimte of the numer of different tx in the smple nd some ide out the evenness of their distriution. Nonetheless, trflp hs ertin inherent limittions, inluding: (1) possile PCR is, preventing the quntifition of the reltive undne in the ommunity (Suzuki & Giovnni 1996), nd (2) the inility to identify different teril speies, euse different teri, regrdless of txon, my produe identil TRFs with given restrition enzyme (Liu et l. 1997). Therefore, flse negtive results my e otined in the unlikely event tht sponge extrt is utilized y ertin teril olonizers whose TRFs re the sme s those produed y the suseptile teri. The present study reveled tht sponge extrts redued the density of enthi ditoms on gel surfes. SIMPER nlysis showed tht the ditoms Nitzshi longissim nd Nviul sp. 1 ounted for lmost 50% of the totl dissimilrity etween ommunities developed on the ontrol nd tretment gels. Similr to the teri, our results on ditoms indite tht sponge metolites ffet the reruitment of ditoms, not only in terms of undne, ut lso qulittively in terms of ommunity struture. After 3 wk, we oserved the presene of mrofouling ommunity, whih ontined 1 lgl nd 5 inverterte speies. The tueworm Hydroides elegns nd the rnle Blnus mphitrite were dominnt in the ommunity. Similr results were oserved in our erlier studies (Qiu et l. 2003). The densities of the mrofoulers were redued in the presene of sponge extrts. We did not find ny differenes in mrofouler density etween extrts of different sponges, whih my hve een used y the low settlement rte during the period of experiments. How do sponge extrts suppress oth miro- nd mrofouling? First, sponge metolites might e toxi to the fouling orgnisms (Beerro et l. 1997, Amsler et l. 2000, Lee & Qin 2003, Doretsov et l. 2004). Compounds exuded y the sponge Aplysin fistulris, ourring in intertidl res, were toxi to gstropod veliger lrve of nudirnhs nd used norml ehvior in ll inverterte dults, s reported y Thompson et l. (1985). The sponge Myle dherens inhiited the lrvl settlement of the tueworm Hydroides elegns y exreting toxi wterorne ompounds (Lee & Qin 2003). In our previous experiments, onditioned sewter from the sponge Cllyspongi pulvint ws toxi to the enthi ditom Nitzshi plee nd to H. elegns lrve (Doretsov et l. 2004). Seond, sponge ompounds might repel propgules of fouling orgnisms. Although repellent properties of sponge extrts hve not yet een desried, repellent tivities of lgl ompounds (Sieurth & Conover 1965, Doretsov 1999) nd teril ompounds (Chet & Mithell 1976, Boyd et l. 1999) hve een identified. A third possiility is tht the hemil ompounds of sponges might ffet miro- nd mrofouling dhesion. Crude extrts of the Crien sponges Ailohroi rss, Chondrill nuult, Etyoplsi ferox nd Iotrohot irotult inhiited the tthment of the terium Virio hrveyi (Kelly et l. 2003). Finlly, sponge metolites might inhiit growth of miro- nd mrofoulers. Inhiition of the growth of ditoms y sponge extrts ws desried erlier in our lortory nd field experiments (Doretsov et l. 2004, 2005). However, the tul mehnisms of the suppression of fouling y sponge ompounds require further investigtion. The present investigtion reveled tht, in most ses, there ws not muh differene in the ntifouling tivity of the MeOH nd DCM extrts of the sponges. This my e so euse most non-polr nd medium-polr ompounds nd orgni slts dissolved suessfully in oth solvents. In the field experiments, only the MeOH extrts of Hlilon sp. nd Cllyspongi sp. hnged the omposition of ditom ommunities, ut the DCM extrts did not initite ny hnges. These oservtions my e explined y the presene of some wter-solule metolites in the MeOH extrts of the sponges, whih my hve ntiditom tivity (Doretsov et l. 2004). In this study, the results of the field experiments with sponge extrts in most ses orroorted the lortory oservtions of epiiosis on the sponge surfes; in some ses, they did not. For exmple, we did not oserve ditoms on the surfes of the sponges, ut found low ditom fouling on the gels with sponge extrts in the field experiments. We oserved high teril density nd high diversity of teri on the surfes of the sponge Hlilon sp., ut extrts of this sponge inhiited formtion of teril iofilms in the field experiments. We suggested severl possile resons for this surprising result. First, the onentrtions of the ompounds used in our experiments were different from those to whih tht teri ws

10 128 Mr Eol Prog Ser 297: , 2005 exposed in nturl onditions. We used the tissue-level onentrtion of sponge metolites, whih ssumes tht there is n equl distriution of sponge ompounds (Jensen et l. 1996). However, the distriution of the ntifouling ompounds in sponges my e different. Moreover, the metolites relesed y extrtion with solvents my not e relesed t ny ppreile onentrtion on the sponge surfe, ut stored inside ells tht hve no effet on surfe iot in the nturl environment. Seondly, ntifouling protetion y the sponges my e result of vrious ntifouling mehnisms, e.g. phgoytosis, slime prodution, prodution of hemil ompounds, nd/or mehnil defenses y skeletl spiules. Thirdly, the high pumping tivity of sponges my use the pssive umultion of teri on the surfe of sponges nd use the high density nd diversity of teri on the surfes of sponges ompred with the surfes of gels with sponge extrts. The present investigtion demonstrted tht sponge metolites my ffet the formtion of miro- nd mrofouling ommunities in situ. The presene of high numers of teri on the surfes of some sponge speies ws lso oserved. At the sme time, our investigtion provided only preliminry informtion out the ntifouling effet of sponge ompounds on fouling ommunities. Further investigtions to eluidte the hemil omposition of sponge-derived ompounds, their mode of tion nd their effetiveness t nturl onentrtion levels re wrrnted. Also, other ntifouling mehnisms, suh s sponge surfe topogrphy, wettility, prodution of slime nd the presene of phgoyti surfe ells, should e onsidered. Aknowledgements. This investigtion ws supported y Hong Kong RGC grnts (HKUST6119/01M, HKUST6100/ 02M, HKUST6240/04M) to P.-Y.Q. The uthors thnk Y. V. Plkhotnikov nd Y. K. Tm (Hong Kong) for ssistne in ommunity fingerprint nlysis. 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