The ISME Journal (2007) 1, 654–659 & 2007 International Society for Microbial Ecology All rights reserved 1751-7362/07 $30.00 www.nature.com/ismej SHORT COMMUNICATION The diversity of the bacterial communities associated with the azooxanthellate hexacoral Cirrhipathes lutkeni Lory Z Santiago-Va´zquez1,5, Thomas B Bru¨ ck1,5, Wolfram M Bru¨ ck2,5, Angela P Duque-Alarco´n1, Peter J McCarthy2 and Russell G Kerr1,3,4 1Department of Chemistry and Biochemistry, Center of Excellence in Biomedical and Marine Biotechnology, Florida Atlantic University, Boca Raton, FL, USA; 2Center for Ocean Exploration, Harbor Branch Oceanographic Institution, Fort Pierce, FL, USA; 3Department of Chemistry, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada and 4Department of Biomedical Sciences, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada This study examined the symbiotic microbiota of the hexacoral Cirrhipathes lutkeni using traditional plate culture, fluorescence in situ hybridization (FISH) and 16S rDNA characterization. FISH counts for the whole coral (holobiont) showed a major presence of c-Proteobacteria (22%) and Actinobacteria (19%), followed by a-Proteobacteria (14%), Firmicutes (9%), Cytophaga-Flavobacter- ium (7%), b-Proteobacteria (6%) and Chloroflexi (2%). In contrast to the diversity observed by FISH, plate cultures were found to be selective for c-Proteobacteria (22 cultures) with the exception of an Actinobacterium. The methods employed in this study detected 76% of all microbes estimated by DAPI staining of C. lutkeni homogenates. The absence of zooxanthellae in this particular hexacoral was confirmed by PCR and spectrophotometry using fresh tissue isolated from the holobiont. This is the first study describing the microbial associations of shallow-water hexacorallia, which opens further insight into coral microbial ecology and may enhance the search for novel natural products in the near future. The ISME Journal (2007) 1, 654–659; doi:10.1038/ismej.2007.77; published online 18 October 2007 Subject Category: microbial population and community ecology Keywords: Cirrhipathes lutkeni; microbiota; culture; FISH Introduction to pathogenic processes, it is important to define microbial associations in healthy individuals. In contrast to studies on sponge microbiota (Webster This study focuses on the bacteria associated with and Hill, 2001; Hentschel et al., 2003; Imhoff and the Western Atlantic black wire-coral Cirrhipathes Sto¨hr, 2003), studies on the bacterial diversity lutkeni (Order: Hexacorallia, Genus: Antipatharia). associated with non-diseased corals are rare. Most Hexacorallia include approximately 200 recognized studies focusing on coral microbiota compare the species of black corals (Echevarrı´a, 2002). Research microbe associations of healthy versus diseased on these organisms, which mostly inhabit deep scleractinians (Bourne and Munn, 2005; Yokouchi water, is limited due to complications associated et al., 2006; Klaus et al., 2007). By contrast, little is with collecting in these environments. Compared to known about the bacterial associations of soft corals, other black corals, C. lutkeni is found at shallower although many bioactive metabolites have been depths (below 30 m); yet very little is known about isolated from these organisms and symbiotic bacter- the biology of this particular coral genus. While ia are thought to be source of these compounds many shallow-water hard and soft corals are known (Fenical 1987; Schmidt et al., 2000; Harder et al., to harbor endosymbiotic dinoflagellates (zoox- 2003; Penn et al., 2006; Bru¨ ck et al., 2007; Webster anthellae, Symbiodinium sp.), it is unknown and Bourne, 2007). As soft corals are also sensitive whether C. lutkeni also requires endophytic sym- bionts for long-term survival (Rosenberg et al., Correspondence: RG Kerr, Department of Chemistry, University of 2007). Prince Edward Island, 550 University Avenue, Charlottetown, To analyze the bacterial communities of C. PEI, Canada C1A 4P3. lutkeni, we used species-specific fluorescent in situ E-mail: [email protected] hybridization (FISH) oligonucleotide probes and 5These authors have made equal contributions to this publication. Received 11 June 2007; revised and accepted 27 August 2007; traditional plate culture along with PCR-based 16S published online 18 October 2007 rRNA gene identification of bacterial isolates (Bru¨ ck Microbial diversity associated with C. lutkeni LZ Santiago-Va´zquez et al 655 et al., 2007). The presence of endophytic symbionts and several species-specific FISH probes according was assessed using dinoflagellate-specific PCR to established protocols (Bru¨ ck et al., 2007). probes and spectrophotometric methods detecting Seawater (3 Â 10 l) was collected and passed dinoflagellate chlorophyll absorbance patterns. through 0.2 mm Nylon filters (Millipore, Bedford, MA, USA). The retentate was fixed on the filter matrix with 100% (v/v) ethanol and used for FISH analysis using the same probes used to analyze the Materials and methods coral. All chemicals were obtained from Sigma Chemical Phylogenetic trees of the 16S rRNA data were Co. (St Louis, MO, USA). Preformulated bacterial constructed using ARB (Ludwig et al., 2004). Novel media were supplied by Difco Laboratories (Detroit, sequences were paired with known sequences from MI, USA). the NCBI database to show their relatedness. Several colonies of C. lutkeni were collected by Sequences are available in GenBank (DQ857736 scuba in June 2005 at the Jim Atria wreck site, through DQ857758). Pompano Beach, Florida at a depth of 45 m (27 1C). Live coral specimens were handled aseptically and Results and discussion homogenized as previously described (Bru¨ ck et al., 2007). Plate cultures were prepared by spreading FISH analysis of C. lutkeni showed that species- dilute coral homogenate on Nutrient (NA) and specific probes covered 76% of the total microbial diluted Marine Agar (MA, 9.2 g Marine Agar 2216, diversity visualized by DAPI staining (Figure 1). 12.5 g Bacto agar, 1 l 83% artificial sea water) and The g- (22%), a-Proteobacteria (14%) and Actino- incubated aerobically at 27 1C (Webster and Hill, bacteria (19%) were the most abundant groups. 2001). Pure cultures were isolated over a 3-month g-Proteobacteria are the most common group in period and sequenced (Bru¨ ck et al., 2007). All other tropical soft-corals (Harder et al., 2003; Bru¨ ck isolates were assessed using Gram stain and motility et al., 2007; Webster and Bourne 2007), whereas (hanging drop technique) to confirm 16S rRNA gene many shallow-water scleractinians feature a-Proteo- sequence identities. Testing for the presence of bacteria as the major group (Yokouchi et al., 2006; dinoflagellate symbionts in coral tissue was carried Klaus et al., 2007). out using standard PCR and spectrophotometric The remaining bacterial groups in C. lutkeni are techniques (Bru¨ ck et al., 2007). Firmicutes (9%), Cytophaga-Flavobacterium (7%), Bacterial counts per sample were estimated with b-Proteobacteria (6%) and Chloroflexi (2%), which DAPI staining as well as with a general eubacterial were also a minor fraction of the bacterial microbiota DAPI EUB388 ALF968 GAM42a BET42a CF319a LGC354suite GNS934 DLP 9 8 7 6 5 LOG cells/g (LOG cells/L) 4 3 Cirrhipathes lutkeni Water Column Figure 1 Log average populations of bacteria associated with C. leutkeni and the surrounding water column estimated by means of FISH and DAPI counting. Error bars are 71 standard deviation. LOG cells per gram of coral is the logarithmic (base 10) cell count (bacteria) per gram of coral (wet weight). LOG cells per liter is the logarithmic (base 10) cell count (bacteria) per liter of water column (wet weight). Probes stated in legend are specific for the following organisms: DAPI: total counts (all DNA present in sample), EUB338: total counts (most bacterial genera), ALF968: a-Proteobacteria, BET42a: b-Proteobacteria, GAM42a: g-Proteobacteria, CF319a: Cytophaga- Flavobacterium, LGC354suite: Firmicutes, GNS934: Chloroflexi, DLP: Actinobacteria. The ISME Journal Microbial diversity associated with C. lutkeni LZ Santiago-Va´zquez et al 656 of other tropical soft corals. Approximately 24% of where 51% of the bacteria were not covered by FISH the organisms detected by DAPI and EUB338 were probes used in the study. The prevalence of not covered by the species-specific FISH probes g-Proteobacteria in both C. lutkeni and octocoral used here. Other studies using different types samples suggests that they may have a symbiotic samples (human and rhesus monkey feces) reported function related to nutrient uptake as was pre- comparable levels of species coverage (approxi- viously observed in Rhopaloeides odorabile and mately 31%–98% detection with probes used) Pocillopora damicornis (Bourne and Munn, 2005). (Bru¨ ck et al., 2006, 2003). It may have been possible The bacterioplankton background from the water to observe a larger number of the total microbiota by column is a problem when assessing the resident FISH with the use of additional probes specific for microbiota of marine species (Hentschel et al., archaea and other organisms. 2003). FISH analysis of the surrounding water However, as was previously observed in other column showed that Actinobacteria (23%) and studies, detection of all unknown organisms a-Proteobacteria (19%) were the major groups while contained in the sample may be impossible by g-Proteobacteria (11%) was less prominent, indicat- existing FISH methods due to the relatively