Microbial Communities Associated with the Invasive Hawaiian Sponge Mycale Armata

The ISME Journal (2009) 3, 374–377 & 2009 International Society for Microbial Ecology All rights reserved 1751-7362/09 $32.00 www.nature.com/ismej SHORT COMMUNICATION Microbial communities associated with the invasive Hawaiian sponge Mycale armata

Guangyi Wang, Sang-Hwal Yoon and Emilie Lefait Department of Oceanography, University of Hawaii at Manoa, Honolulu, HI, USA

Microbial symbionts are fundamentally important to their host ecology, but microbial communities of invasive marine species remain largely unexplored. Clone libraries and Denaturing gradient gel electrophoresis analyses revealed diverse microbial phylotypes in the invasive marine sponge Mycale armata. Phylotypes were related to eight phyla: Proteobacteria, Actinobacteria, Bacter- oidetes, Cyanobacteria, Acidobacteria, Chloroflexi, Crenarchaeota and Firmicutes, with predominant alphaproteobacterial sequences (458%). Three Bacterial Phylotype Groups (BPG1–– associated only with sequence from marine sponges; BPG2––associated with sponges and other marine organisms and BPG3––potential new phylotypes) were identified in M. armata. The operational taxonomic units (OTU) of cluster BPG2-B, belonging to Rhodobacteraceae, are dominant sequences of two clone libraries of M. armata, but constitute only a small fraction of sequences from the non-invasive sponge Dysidea sp. Six OTUs from M. armata were potential new phylotypes because of their low sequence identity with their reference sequences. Our results suggest that M. armata harbors both sponge-specific phylotypes and bacterial phylotypes from other marine organisms. The ISME Journal (2009) 3, 374–377; doi:10.1038/ismej.2008.107; published online 6 November 2008 Subject Category: microbial ecology and functional diversity of natural habitats Keywords: sponge–microbe symbiosis; invasive marine sponges; microbial diversity; Mycale armata

Marine invasive species pose a serious threat to the library construction and denaturing gradient gel world’s oceans. Symbiotic microbes can dramati- electrophoresis analysis (Gao et al., 2008; Zhu et al., cally impact ecosystem function by changing the 2008). Band patterns of microbial communities phenotype of their hosts (Gerardo et al., 2006). varied in different sponge species collected at the However, microbial communities within invasive same time, but bands were similar in samples of the marine species remain relatively unknown. The same sponge species collected at different times marine sponge Mycale armata (family Mycalidae) (Figure 1). Seven major bands were consistently is a recent, unintentionally introduced sponge present in samples of M. armata collected in both species, becoming invasive and the most abundant years. Two bands (1 and 2), which were common to sponge species in the Hawaiian reef ecosystem. In M. armata and other non-invasive sponges (Dysidea Kaneohe Bay (Oahu, Hawaii), this species overgrows sp., Gelliodes fibrosa, Tedania sp. and Scopalina and kills native coral lagoon–patch reef commu- sp.), and two other, unique bands (3 and 4) were nities (Supplementary Figure 1). To reveal microbial detected in the samples of M. armata collected in features of M. armata, molecular methods were both years. The denaturing gradient gel electrophor- applied to investigate its microbial communities. esis band patterns suggested that M. armata con- Sponge samples were collected along the shores of tained microbial phylotypes present in the other Coconut Island in August 2004 and May 2005, Hawaiian sponges, but also harbored unique micro- situated within Kaneohe Bay and surrounded by 64 bial phylotypes and these phylotypes seemed to acres of coral reef. Samples of sea water and sponges have little temporal changes. (invasive and non-invasive) were collected and To reveal microbial composition in M. armata, processed according to the method of Gao et al. four clone libraries were constructed from the (2008). Total genomic DNA was used as a PCR samples of M. armata (A and B for samples collected template for the amplification of 16S rRNA genes for in August 2004 and May 2005, respectively), Dysidea sp. (C) and sea water (D). Clone sequences were de-replicated first and then grouped into the Correspondence: G Wang, Department of Oceanography, Univer- same operational taxonomic unit (OTU) using a sity of Hawaii at Manoa, 1000 Pope Road, MSB205, Honolulu, percent sequence identity of greater than 97%. HI 96822, USA. E-mail: [email protected] The percentage coverage and diversity of the four Received 1 September 2008; revised 2 October 2008; accepted 8 libraries are summarized in Supplementary Table 1. October 2008; published online 6 November 2008 Analysis of clone sequences identified 24 and 19 Microbial diversity of an invasive marine sponge G Wang et al 375 ABCDEFG Three Bacterial Phylotype Groups (BPGs) were identified from OTUs derived from M. armata and Dysidea sp. (Figure 2). The first group (BPG1) included 10 OTUs affiliated only with sponge- derived 16S rRNA bacterial sequences. Of this group, three OTUs of cluster BPG1-D were members of Gammaproteobacteria and found in the samples 1 of M. armata collected in both years. Three OTUs of clade BPG1-B were closely clustered with betapro- 3 teobacterial sequences identified in Mediterranean 4 sponge Tethya aurantium (Thiel et al., 2007b) and the Antarctic sponges Latrunculia apicalis and M. acerata (Webster et al., 2004). The OTUs of two 2 clusters BPG1-C and BPG1-D (Figure 2) were phylogenetically related to gammaproteobacterial sequences from the Hawaiian sponges Suberites zeteki (Zhu et al., 2008) and the Great Barrier Reef sponge Rhopaloeides odorabile (Webster et al., 2001), respectively. Figure 1 Denaturing gradient gel electrophoresis (DGGE) band- The second group (BPG2) contained 14 OTUs ing profiles of bacterial 16S rRNA genes PCR-amplified from total DNA extracted from sponges using the primers GC-BAC341 and related to 16S rRNA bacterial sequences from both BAC521. Lanes A and G, Dysidea sp.; lane C, Gelliodes fibrosa; sponges and other marine organisms, such as corals lanes B and D, Mycale armata; lane E, Tedania sp. and lane F, and squids. Seven OTUs of cluster BPG2-B belonged Scopalina sp. Lanes D–G, samples collected at Kaneohe bay in to Alphaproteobacteria and were present in all August 2004. Lanes A–C, samples collected in May 2005 from the same location. PCR products were concentrated and prepared as samples of M. armata and Dysidea sp. (Figure 2). described by Gao et al. (2008). These OTUs were predominant among clone sequences from two M. armata libraries, but contrib- uted only to a small fraction of clone sequences from OTUs from M. armata libraries A and B, respec- Dysidea sp. The OTUs of clusters BPG2-C and BPG2- tively. Additional 27 and 15 OTUs were identified E were members of Alpha- and Gammaproteobacter- from clone libraries of Dysidea sp. (C) and sea water ia, respectively, and were found in the samples of M. (D), respectively. Sequences of 16S rRNA gene armata collected in both years (Figure 2). Interest- clones from M. armata libraries were related to ingly, two OTUs (MA17 and MB73) of cluster BPG2- members of Proteobacteria (classes Alpha-, Beta-, C were closely related to Pseudovibrio denitrificans Delta- and Gammaproteobacteria), Bacteroidetes, isolated from sea water in Taiwan (Shieh et al., Actinobacteria, Cyanobacteria, Firmicutes, Chloro- 2004) and to the P. denitrificans-like clone se- flexi, Acidobacteria and Crenarchaeota (Figure 2; quences from 11 marine sponges (Enticknap et al., Supplementary Figures 2 and 3). Alphaproteobac- 2006; Hentschel et al., 2006; Taylor et al., 2007) with teria were a dominant microbial group, comprising 98% and 99% sequence identity, respectively, in 61.3% and 58.0% of clone sequences from libraries 16S rRNA gene sequences. Because of potential A and B, respectively. Although alphaproteobacter- function of the P. denitrificans-like phylotypes in ial sequences have been observed in 16S rRNA gene host nitrogen metabolism (Webster and Hill, 2001; libraries constructed from over 12 marine sponges Enticknap et al., 2006), these OTUs are a promising collected from several ocean provinces (Hentschel target for future exploration of their physiological et al., 2006; Taylor et al., 2007; Thiel et al., 2007a, b; function in M. armata. The last group (BPG3), Zhu et al., 2008), this is only the second report of including six OTUs, comprised potentially new alphaproteobacterial dominance in the 16S rRNA bacterial phylotypes because they branched into clone library, after the report on Halichondria new clusters, without immediate phylogenetic panicea (Wichels et al., 2006). The three classes neighbors (for example, MA47 and MA43). The (Alpha-, Gamma- and Deltaproteobacteria) and three remaining OTUs were the nonspecific sequences phyla (Actinobacteria, Cyanobacteria and Bacteroi- because of their phylogenetic affiliation with 16S detes) were found in both M. armata clone libraries rRNA gene sequences from sea water, sediments and (A and B). Sequences of chloroplasts and the other environments (for example, MB12 and DC31). phylum Verrucomicrobia were found exclusively Finally, most OTUs of Bacteroidetes, Acidobacter- in Dysidea sp. whereas members of Actinobacteria, ia, Firmicutes, Actinobacteria, Chloroflexi and Cre- Crenarchaeota and Deltaproteobacteria were present narchaeota from M. armata and Dysidea sp. only in one (A or B) library of M. armata (Figure 2). displayed close phylogenetic affiliations with se- Fewer phyla were identified in the seawater library, quences derived from sea water or sediments, but unclassified bacterial and euryarchaeote se- instead of from marine sponges (Supplementary quences (Supplementary Figure 2) were present Figure 2). None of the cyanobacterial sequences only in the seawater library. from M. armata and Dysidea sp. were related to the

The ISME Journal Microbial diversity of an invasive marine sponge G Wang et al 376 100 sponge clone DC1 BPG2-A 75 coral Rhodospirillaceae bacterium clone BME47, DQ917824 86 sponge clone TAA-10-03, AM259856 (Tethya aurantium) marine worm endosymbiont clone SpecB-al-16, AJ890096 92 sponge clone MA40 BPG1-A 100 65 sponge clone PR11, DQ904006 (Suberites zeteki) 72 sponge clone PR1, DQ904005 (Suberites zeteki) 84 100 sponge clone Ax36-F7, EF092208 (Axinella corrugata) sponge clone MAx29-GX, EF092257 (Axinella corrugata) 100 sponge clone MA47 BPG3-A 98 sponge clone MB42 100 octacoral clone NISA063, DQ396195 sponge bacterium Sphingopyxis sp. isolate MOLA516, AM990740 (Petrosia ficiformis) 51 seawater bacterium clone DR55oSWSAEE43, DQ354731 sponge clone MA34 BPG2-B sponge clone DC16 sponge clone MB18 sponge clone MB16 100 sponge clone MA51 sponge clone MB37 56 sponge clone DC13 marine squid bacterium clone T62ANG12, AJ63954 66 50 black band diseased coral tissue clone F1_32f, EF123406 sponge Rhodobacteraceae bacterium N06ML5, EF629850 (Mycale laxissima) coral bacterium isolate slcb31, DQ416551 84 coral bacterium clone BBD_217_35, DQ446160 -proteobacterium 100 squid bacterium clone 6ANG58B, AJ633976 sponge bacterium isolate 1109, DQ888839 (Tethya aurantia) 69 sponge bacterium isolate D29, DQ227659 (Pseudoceratina clavata) 70 sponge clone MA74 85 seawater bacterium clone D2578, EU259793 seawater bacterium clone EV818CFSSAHH36, DQ336987 61 78 sponge clone MA17 BPG2-C sponge clone MB73 sponge -proteobacterium isolate JE062, DQ097238 (Mycale laxissima) sponge Pseudovibrio sp. clone AC2-A5, EF092159 (Axinella corrugata) 100 sponge -proteobacterium isolate JE066, DQ097263 (Microciona prolifera) sponge -proteobacterium isolate JE013, DQ097253 (Acanthostronglyophora sp.) sponge -proteobacterium isolate JE008, DQ097248 (Didiscus oxeata) sponge -proteobacterium isolate JE005, DQ097246 (Monanchora unguifera) coral (Oculina patagonica) bacterium isolate slcb33, DQ416557 sponge -proteobacterium MBIC3368, AB012864 (Unknown sponges) 96 sponge clone DC5 BPG3-B 63 sponge clone DC22 87 squid (Photololigo chinensis) -proteobacterium clone 29ANG4, AJ633952 88 sponge clone DC21 BPG2-D squid (Photololigo duvaucelii) -proteobacterium clone T63ANG236, AJ633963 99 sponge clone MB14 BPG1-B sponge clone DC27 sponge clone DC11 87 sponge bacterium clone L44, AY321402 (Latrunculia apicalis) 100 sponge bacterium clone L8, AY321404 (Latrunculia apicalis) sponge bacterium clone M23, AY321412 (Mycale acerata) 100 sponge bacterium clone L9, AY321404 (Latrunculia apicalis) β-proteobacterium 51 sponge bacterium clone TAA-5-58, AM259836 (Tethya aurantium) sponge proteobacterium clone TAA-5-43, AM259835 (Tethya aurantium) 90 sponge bacterium clone TAI-2-47, AM259770 (Tethya aurantium) 100 sponge bacterium clone TAI-2-157, AM259777 (Tethya aurantium) sponge bacterium clone TAA-10-30, AM259832 (Tethya aurantium) sponge proteobacterium clone TAI-2-47, AM259770 (Tethya aurantium) sponge clone MA43 bacterium clone RL183_aao01f06, DQ800655 61 sponge clone MA77 Shigella sp. clone 284b, EU644458 sponge clone DC4 99 85 sponge clone DC30 Vibrio natriegens strain V6, EU636230 60 100 76 Vibrio parahaemolyticus isoatate, DQ026024 81 sponge Vibrio sp. clone Met1, DQ904002 (Suberites zeteki) sponge Vibrio sp. clone Met5, DQ904003 (Suberites zeteki) 87 98 sponge Vibrio sp. clone Met18, DQ904004 (Suberites zeteki) sponge clone MB4A BPG1-C 86 sponge clone DC35A 98 sponge clone MA49 BPG2-E 65 100 sponge clone MB1 coral Pseudoalteromonas sp. isolate L-fcr-129, EU420061 100 coral Pseudoalteromonas sp. isolate Cobs2H204, EU246846 -proteobacterium shrimp Pseudoalteromonas sp. isolate RW19, EU419906 99 sponge clone MA67 100 hydrothermal -proteobacterium clone Pitb-vmat-87, AB294978 91 sponge clone MA60 97 sediment bacterium clone Fitz2-15, DQ256675 74 sediment bacterium clone S26-65, EU287365 sponge bacterium clone NWCu007, AF313496 (Rhopaloeides odorabile) 100 sponge clone MA19 BPG1-D 100 sponge clone MB5 sponge clone MA21 100 mollusk Endozoicomonas elysicola isolate MKT110, AB196667 sponge -proteobacterium clone CN34, AM259915 (Chondrilla nucula) 63 sponge -proteobacterium clone HAL-T-6, EU350863 (Haliclona simulans) sponge -proteobacterium clone HOC2, AB054136 (Halichondria okadai) 100 sponge -proteobacterium clone MOLA531, AM990755 (Petrosia ficiformis) soft coral Spongiobacter sp. clone ME19, DQ917863 60 Spongiobacter nickelotolerans, AB205011 (unknown marine sponges) Acidobacterium clone AKYG1769, AY921986 0.1 substitutions/site

Figure 2 Neighbor-joining phylogenetic tree of proteobacterial 16S rRNA gene sequences derived from the Hawaiian sponges Mycale armata and Dysidea sp. Clone sequences from the 16S rRNA clone libraries A and B for M. armata, library C for Dysidea sp., and library D for sea water are designated with the prefix MA, MB, DC and SW, respectively, and shown in bold. Libraries A, C, and D were constructed from the samples of M. armata, Dysidea sp. and sea water, respectively, were collected in August 2004; library B constructed from the samples of M. armata were collected in May 2005. For matched sequences derived from sponges, the sponge species name is given in parentheses following the sequence accession number. Shaded sequences are OTUs belonging to one of three bacterial phylotype groups (BPG1, BPG2 and BPG3). Numbers above branches indicate bootstrap values of neighbor-joining analysis (450%) from 1000 replicates.

sponge-specific cyanobacterium Candidatus Syne- species, such as scleractinian corals, pogonophoran chococcus spongiarum (Usher et al., 2004) (Supple- and lucinid bivalves, to lead partially or exclusively mentary Figure 3). autotrophic lives in tropical reefs, sulfur-rich habi- Invertebrate endosymbioses have major ecological tats and other marine environments (Saffo, 1992). impacts both on habitat range and on the ecosystems Sponge symbionts are thought to benefit their hosts of which they are a part. Symbioses with auto- in many ways (Wang, 2006; Taylor et al., 2007). This trophic symbionts have allowed several marine study revealed the presence of potentially new

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