Development of Bacterial Biofilms on Artificial Corals in Comparison to Surface-Associated Microbes of Hard Corals

Numerous studies have demonstrated the differences in bacterial communities associated with corals versus those in their surrounding environment. However, these environmental samples often represent vastly different microbial micro-environments with few studies having looked at the settlement and growth of bacteria on surfaces similar to corals. As a result, it is difficult to determine which bacteria are associated specifically with coral tissue surfaces. In this study, early stages of passive settlement from the water column to artificial coral surfaces (formation of a biofilm) were assessed. Changes in bacterial diversity (16S rRNA gene), were studied on artificially created resin nubbins that were modelled from the skeleton of the reef building coral Acropora muricata. These models were dip-coated in sterile agar, mounted in situ on the reef and followed over time to monitor bacterial community succession. The bacterial community forming the biofilms remained significantly different (R = 0.864 p<0.05) from that of the water column and from the surface mucus layer (SML) of the coral at all times from 30 min to 96 h. The water column was dominated by members of the α-proteobacteria, the developed community on the biofilms dominated by γ-proteobacteria, whereas that within the SML was composed of a more diverse array of groups. Bacterial communities present within the SML do not appear to arise from passive settlement from the water column, but instead appear to have become established through a selection process. This selection process was shown to be dependent on some aspects of the physico-chemical structure of the settlement surface, since agar-coated slides showed distinct communities to coral-shaped surfaces. However, no significant differences were found between different surface coatings, including plain agar and agar enhanced with coral mucus exudates. Therefore future work should consider physico-chemical surface properties as factors governing change in microbial diversity

Variation in 16S rRNA gene fingerprints between sample types (Biofilm, SML and water column), for March 2009 (summer); (a) Composite DGGE image standardised for gel-to-gel comparison using BioNumerics, (b) Multidimensional scaling (MDS) plot based on relative band intensity from composite DGGE profile.

<p>Variation in 16S rRNA gene fingerprints between sample types (Biofilm, SML and water column), for March 2009 (summer); (a) Composite DGGE image standardised for gel-to-gel comparison using BioNumerics, (b) Multidimensional scaling (MDS) plot based on relative band intensity from composite DGGE profile.</p

Variation in 16S rRNA gene fingerprints between sample types (spatial samples A–E) for summer season (March 2009); (a) Composite DGGE image standardised for gel-to-gel comparison using BioNumerics, (b) Multidimensional scaling (MDS) plot based on relative band intensity from composite DGGE profile of the biofilm.

<p>Variation in 16S rRNA gene fingerprints between sample types (spatial samples A–E) for summer season (March 2009); (a) Composite DGGE image standardised for gel-to-gel comparison using BioNumerics, (b) Multidimensional scaling (MDS) plot based on relative band intensity from composite DGGE profile of the biofilm.</p

Box-plot showing Shannon-Weiner diversity index of the SML samples of <i>Acropora muricata</i> taken over four consecutive days, based on DGGE 16S rRNA gene diversity compared to that of the water column (WC).

<p>Box-plot showing Shannon-Weiner diversity index of the SML samples of <i>Acropora muricata</i> taken over four consecutive days, based on DGGE 16S rRNA gene diversity compared to that of the water column (WC).</p

Composite DGGE image showing replicates collected at 4 h of biofilm development on microscopic slides and replica coral nubbins with dominant bands sequenced (<b>Table 2</b>), gel-to-gel comparisons were standardised using internally run marker lanes and analysed using BioNumerics software.

<p>S =  number of bands visible in DGGE using BioNumerics representing relative diversity.</p

Multidimensional scaling plot (MDS), showing seasonal changes in bacterial communities (16S rRNA gene fingerprints), developing on the biofilm of the replica coral nubbins enriched with agar; (a) average of n = 3 replicates for different time scales of biofilm development for both seasons; summer (s) (March 2009) and winter (w) (August 2008), (b–h) representatives of the sequenced ribotypes responsible for the greatest differences between seasons, Latin name and gen bank sequence ID included.

<p>Size of bubble depicts intensity of band/ribotype on DGGE within individual samples.</p

Example of novel methodology utilised in this study and site location; a) Photograph of replica coral nubbins used in experiment with close up sections of the mould (insets), b) Site map showing, Heron Island GBR, Australia (23°27′S, 151°55′E), location of main study site (A) the Reef Flat and those used in spatial sampling; (B) Coral Gardens 23°26.839/151°54.717 (C) Lagoon 23°27.272/151°57.921 (D) 3^rd/4^th Point 23°26.146/151°58.833 (E) Wistari 23°29.081/151°54.015.

<p>Arrows depict water current direction at time of sampling with direction and speed noted. Samples were taken on calm days, one hour before high tide, with wave speed W_S<0.5 m/s and wave heights H_S<0.5 m. Scale bar  = 1 km.</p

Shannon-Weiner diversity based on DGGE composite (a) Box-plots of the winter season (August 2008), (b) DGGE composite image (winter), (c) box plot of summer season (March 2009), (d) DGGE composite image (summer).

<p>Arrow depicts storm event with increased chop (winds above 35 km/h). Average wind speed for other sample periods was below 20 km/h. WC  =  water column.</p

Table showing the dominant 16S rRNA gene ribotypes, explaining the greatest differences/similarities between samples, excised from the DGGE gel.

<p>Representatives from each sample types were included; (Biofilm [agar slides], Biofilm [agar coated artificial nubbins], coral mucus and the water column). Close matches (Blast nt), species identification, group affiliation (identified to closest published relatives on GenBank at the time of comparison) are included within the table. All samples were collected from Heron Island reef flat, March 2009.</p