Introduction

In contrast to studies on sponge microbiota (Webster and Hill, 2001; Hentschel et al., 2003; Imhoff and Stöhr, 2003), studies on the bacterial diversity associated with non-diseased corals are rare. Most studies focusing on coral microbiota compare the microbe associations of healthy versus diseased scleractinians (Bourne and Munn, 2005; Yokouchi et al., 2006; Klaus et al., 2007). By contrast, little is known about the bacterial associations of soft corals, although many bioactive metabolites have been isolated from these organisms and symbiotic bacteria are thought to be source of these compounds (Fenical 1987; Schmidt et al., 2000; Harder et al., 2003; Penn et al., 2006; Brück et al., 2007; Webster and Bourne, 2007). As soft corals are also sensitive to pathogenic processes, it is important to define microbial associations in healthy individuals.

This study focuses on the bacteria associated with the Western Atlantic black wire-coral Cirrhipathes lutkeni (Order: Hexacorallia, Genus: Antipatharia). Hexacorallia include approximately 200 recognized species of black corals (Echevarría, 2002). Research on these organisms, which mostly inhabit deep water, is limited due to complications associated with collecting in these environments. Compared to other black corals, C. lutkeni is found at shallower depths (below 30 m); yet very little is known about the biology of this particular coral genus. While many shallow-water hard and soft corals are known to harbor endosymbiotic dinoflagellates (zooxanthellae, Symbiodinium sp.), it is unknown whether C. lutkeni also requires endophytic symbionts for long-term survival (Rosenberg et al., 2007).

To analyze the bacterial communities of C. lutkeni, we used species-specific fluorescent in situ hybridization (FISH) oligonucleotide probes and traditional plate culture along with PCR-based 16S rRNA gene identification of bacterial isolates (Brück et al., 2007). The presence of endophytic symbionts was assessed using dinoflagellate-specific PCR probes and spectrophotometric methods detecting dinoflagellate chlorophyll absorbance patterns.

Materials and methods

All chemicals were obtained from Sigma Chemical Co. (St Louis, MO, USA). Preformulated bacterial media were supplied by Difco Laboratories (Detroit, MI, USA).

Several colonies of C. lutkeni were collected by scuba in June 2005 at the Jim Atria wreck site, Pompano Beach, Florida at a depth of 45 m (27 °C). Live coral specimens were handled aseptically and homogenized as previously described (Brück et al., 2007). Plate cultures were prepared by spreading dilute coral homogenate on Nutrient (NA) and diluted Marine Agar (MA, 9.2 g Marine Agar 2216, 12.5 g Bacto agar, 1 l 83% artificial sea water) and incubated aerobically at 27 °C (Webster and Hill, 2001). Pure cultures were isolated over a 3-month period and sequenced (Brück et al., 2007). All isolates were assessed using Gram stain and motility (hanging drop technique) to confirm 16S rRNA gene sequence identities. Testing for the presence of dinoflagellate symbionts in coral tissue was carried out using standard PCR and spectrophotometric techniques (Brück et al., 2007).

Bacterial counts per sample were estimated with DAPI staining as well as with a general eubacterial and several species-specific FISH probes according to established protocols (Brück et al., 2007).

Seawater (3 × 10 l) was collected and passed through 0.2 μm 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 coral.

Phylogenetic trees of the 16S rRNA data were constructed using ARB (Ludwig et al., 2004). Novel sequences were paired with known sequences from the NCBI database to show their relatedness. Sequences are available in GenBank (DQ857736 through DQ857758).

Results and discussion

FISH analysis of C. lutkeni showed that species-specific probes covered 76% of the total microbial diversity visualized by DAPI staining (Figure 1). The γ- (22%), α-Proteobacteria (14%) and Actinobacteria (19%) were the most abundant groups. γ-Proteobacteria are the most common group in other tropical soft-corals (Harder et al., 2003; Brück et al., 2007; Webster and Bourne 2007), whereas many shallow-water scleractinians feature α-Proteobacteria as the major group (Yokouchi et al., 2006; Klaus et al., 2007).

Figure 1
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 ±1 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: α-Proteobacteria, BET42a: β-Proteobacteria, GAM42a: γ-Proteobacteria, CF319a: Cytophaga-Flavobacterium, LGC354suite: Firmicutes, GNS934: Chloroflexi, DLP: Actinobacteria.

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The remaining bacterial groups in C. lutkeni are Firmicutes (9%), Cytophaga-Flavobacterium (7%), β-Proteobacteria (6%) and Chloroflexi (2%), which were also a minor fraction of the bacterial microbiota of other tropical soft corals. Approximately 24% of the organisms detected by DAPI and EUB338 were not covered by the species-specific FISH probes used here. Other studies using different types samples (human and rhesus monkey feces) reported comparable levels of species coverage (approximately 31%–98% detection with probes used) (Brück et al., 2006, 2003). It may have been possible to observe a larger number of the total microbiota by FISH with the use of additional probes specific for archaea and other organisms.

However, as was previously observed in other studies, detection of all unknown organisms contained in the sample may be impossible by existing FISH methods due to the relatively high detection limit of approximately 103–104 target cells per ml (Loy et al., 2002).

In comparison, previously examined octocoral homogenates indicated that α-, β- and γ-Proteobacteria were the most common bacterial genera observed (Brück et al., 2007). Actinobacteria were only a minor component of the microbiota (1%–3%). Again, a large number of the bacteria detected by either DAPI or EUB338 were not covered by the species-specific FISH probes. This was particularly prevalent in homogenates of Iciligorgia schrammi where 51% of the bacteria were not covered by FISH probes used in the study. The prevalence of γ-Proteobacteria in both C. lutkeni and octocoral samples suggests that they may have a symbiotic function related to nutrient uptake as was previously observed in Rhopaloeides odorabile and Pocillopora damicornis (Bourne and Munn, 2005). The bacterioplankton background from the water column is a problem when assessing the resident microbiota of marine species (Hentschel et al., 2003). FISH analysis of the surrounding water column showed that Actinobacteria (23%) and α-Proteobacteria (19%) were the major groups while γ-Proteobacteria (11%) was less prominent, indicating a different species distribution in the water sample compared to the C. lutkeni sample set (Figure 1).

Bacterial cultures resulted in 23 distinct bacterial isolates, almost exclusively γ-Proteobacteria (Figure 2), suggesting high selectivity of culture conditions for this group. Culture conditions can influence the distribution of phylogenetic groups in microbial isolates (Imhoff and Stöhr, 2003). Additional information on bacterial characterization can be found in Table 1. The species distribution of C. lutkeni bacterial isolates resembled those of tropical octocorals previously isolated under equivalent culture conditions, which inferred formation of region-specific microbial associations (Brück et al., 2007). Most isolates belonged to the Pseudomonaceae which have been previously associated with marine sediment, octocorallia, scleractinians and the water column (Suzuki et al., 1997; Bourne and Munn, 2005; Penn et al., 2006; Brück et al., 2007). Even though the two isolates of Halomonas sp. and Cobetia sp. detected here were of low sequence homology (94% and 96%, respectively), it may be inferred that their presence appears to be specific to C. lutkeni. The abundance of Halomonadaceae was also reported in deep-water black coral (Penn et al., 2006), which may indicate a species-specific microbial association (Hentschel et al., 2003). It is interesting to note that while most organisms found here are common in an marine environment, Vibrio sp. have often been associated with a variety of coral diseases (Knowlton and Rohwer, 2003; Cervino et al., 2004; Bourne and Munn, 2005). However, no pathological changes were observed in C. lutkeni, indicating that the Vibrio sp. may be part of the natural microbiota. The single isolate belonging to the Actinobacteria, Proprionibacterium sp., has never been isolated from a marine source and based on its low sequence similarity (92%) to other GenBank deposits, may actually represent a new genus of Actinobacteria. Also of note is the identification of Acinetobacter johnsonii in association with this coral. Although this genus is commonly found in terrestrial and oil-polluted marine environments and in association with marine invertebrates (Alvarez et al., 1997; Juni, 2005; Sfanos et al., 2005), A. johnsonii is typically found in clinical specimens, activated sludge and foodstuffs (Juni, 2005). There has been one study in which this bacterium was found in association with a marine sponge (Li et al., 2006); however, further work will be needed to show whether this bacterium is a natural part of the bacterial assemblage of marine invertebrates or whether its presence is due to pollution of the marine environment.

Figure 2
figure 2

De novo phylogenetic tree of culturable bacteria isolated from C. lutkeni. Numbers at nodes are percentages indicating levels of bootstrap support, based on neighbor-joining analysis of 1000 re-sampled data sets. Reference sequences derived from previous studies and GenBank entries are described in the text or are written with their corresponding accession numbers.

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Table 1 Cirrhipathes lutkeni isolates
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The presence of symbiotic dinoflagellates in C. lutkeni was assessed by PCR and spectrophotometric methods. Dinoflagellate-specific chlorophyll (a and c2) absorbance patterns and PCR signals could only be detected in the positive control, the zooxanthellate octocoral Pseudopterogorgia elisabethae, but not in the C. lutkeni sample. The lack of symbiotic dinoflagellates in C. lutkeni (data not shown) is consistent with previous observations that certain species of octo- and hexacorals, which thrive in light-deprived environments, do not require algal symbionts for survival (Penn et al., 2006; Brück et al., 2007).

To the best of our knowledge, this is the first study examining the microbial associations of moderate depth hexacorals (40–100 m). This study provides a preliminary assessment of the microbial ecology of hexacoral-specific microbial associations. Evaluation of microbial isolates for the production of novel bioactive metabolites is currently underway in our laboratory. As Halomonas sp. are excellent producers of poly-hydroxyalkanoates, the novel isolates may also be of interest for the industrial production of biopolymers.