Bacterial communities of two ubiquitous Great Barrier Reef corals reveals both site- and species-specificity of common bacterial associates

Abstract

Background: Coral-associated bacteria are increasingly considered to be important in coral health, and altered bacterial community structures have been linked to both coral disease and bleaching. Despite this, assessments of bacterial communities on corals rarely apply sufficient replication to adequately describe the natural variability. Replicated data such as these are crucial in determining potential roles of bacteria on coral.

Methodology/principal findings: Denaturing Gradient Gel Electrophoresis (DGGE) of the V3 region of the 16S ribosomal DNA was used in a highly replicated approach to analyse bacterial communities on both healthy and diseased corals. Although site-specific variations in the bacterial communities of healthy corals were present, host species-specific bacterial associates within a distinct cluster of gamma-proteobacteria could be identified, which are potentially linked to coral health. Corals affected by "White Syndrome" (WS) underwent pronounced changes in their bacterial communities in comparison to healthy colonies. However, the community structure and bacterial ribotypes identified in diseased corals did not support the previously suggested theory of a bacterial pathogen as the causative agent of the syndrome.

Conclusions/significance: This is the first study to employ large numbers of replicated samples to assess the bacterial communities of healthy and diseased corals, and the first culture-independent assessment of bacterial communities on WS affected Acroporid corals on the GBR. Results indicate that a minimum of 6 replicate samples are required in order to draw inferences on species, spatial or health-related changes in community composition, as a set of clearly distinct bacterial community profiles exist in healthy corals. Coral bacterial communities may be both site and species specific. Furthermore, a cluster of gamma-proteobacterial ribotypes may represent a group of specific common coral and marine invertebrate associates. Finally, the results did not support the contention that a single bacterial pathogen may be the causative agent of WS Acroporids on the GBR.

Figure 1. Study sites.

Study area and sites in relation to region (A), southern Great Barrier Reef (B), Heron Island and Wistari Reef (C). D–F are representative images of corals sampled: apparently healthy Acropora hyacinthus (D), Acropora hyacinthus affected by White Syndrome (E) and apparently healthy Stylophora pistillata. Images are from NASA/Goddard Space Flight Centre Scientific Visualization Studio, E. Sampayo and G. Roff.

Figure 2. Representative denaturing gradient gel electrophoresis (DGGE) gels of bacterial community profiles in healthy corals and water.

DGGE gels of the 16S rDNA hypervariable region V3 show the various bacterial communities present colonies of (A) Stylophora pistillata and (B) Acropora hyacinthus. Water samples and representative profiles of all profile types from all sites (Wistari Reef, Tenements, Harry's Bommie) are included on both gels. Sample numbers are shown above each lane and band numbers indicate the position of bands excised for sequence analyses. *, #, ^∧, § identify samples specifically included because they show significant deviations from the most commonly observed bacterial communities as seen in the majority of samples.

Figure 3. Principal components analyses (PCA) of coral bacterial communities.

PCA of (A) bacterial community diversity in Acropora hyacinthus and Stylophora pistillata, (B) A. hyacinthus bacterial community diversity by site, (C) S. pistillata bacterial community diversity by site. Variation in the community structure explained by the first two PCA axes for A, B and C are 36.4%, 30.3% and 33.9%, respectively. (D, E) Species accumulation curves of A. hyacinthus (D) and S. pistillata (E) plotted for all three sites. Dotted lines represent the estimated curve based on an infinite number of samples (UGE) and the solid lines represent species accumulation based on true values (SobS). Species diversity indices (Shannon-Weaver H′) are further shown for each site. Sites: Wistari = Wistari Reef, Tenements = Tenements, Harry's = Harry's Bommie.

Figure 4. Phylogenetic analyses of obtained sequences and coral associated bacteria.

Maximum likelihood consensus tree of all 16S rDNA V3 sequences obtained from denaturing gradient gel (DGGE) analyses of bacterial communities associated with Stylophora pistillata, Acropora hyacinthus and water samples. For all identified and sequenced DGGE bands, the bacterial group affiliation is shown and the taxonomic identification given to the family level where known (see Tables S1 and S2 for closest relatives). Bootstrap values were calculated based on the 1000 replicate sample sets only of the initial full length sequences used to construct the tree and only values of >50% are shown. Scale bar represents 0.1 substitutions per nucleotide position. Sequences obtained from: GenBank (black); water samples (blue); apparently healthy S. pistillata (red); apparently healthy A. hyacinthus (green), and; diseased A. hyacinthus (green*). GenBank accession numbers GQ924692–GQ924753 for sequences obtained in this study.

Figure 5. Average percent contribution of specific bacterial groups.

Average percent contribution (based on SIMPER analyses results, Table S2) within Stylophora pistillata and Acropora hyacinthus at all three sites (Harry's Bommie, Tenements, Wistari Reef). γ-proteobacteria (including Type A associates, Enterobacteraceae, Pseudomonadaceae, Vibrionaceae) are the main constituents of the bacterial community in both species. Uncharacterised = DGGE bands from which no sequence identity was obtained were grouped; each independently contributed only a small percentage compared to the identified bands (Table S2).

Figure 6. Denaturing gradient gel electrophoresis (DGGE) gels of healthy and diseased corals and water.

DGGE gels of the 16S rDNA hypervariable region V3 show the bacterial communities present in White Syndrome (WS) affected (diseased) Acropora hyacinthus colonies from (A) Wistari Reef and (B) Tenements. The most common healthy A. hyacinthus and water sample DGGE profiles at each site are included as a reference. Sample numbers are shown above each lane and band numbers indicate the position of bands excised for sequence analyses. HP = healthy part of diseased colony 10 cm away from the lesion boundary; LB = tissue from the border of lesion boundary of colony (see Methods for more information). (C) Principal components analyses (PCA) of the bacterial community diversity in apparently healthy and WS diseased A. hyacinthus. ^∧ ,§ identify apparently healthy samples from Figure 2, which are more similar to ‘diseased’ (WS) samples than they are to other apparently healthy samples. Variation in the community structure explained by the first two PCA axes is 27.3%.