Metamorphosis of a scleractinian coral in response to microbial biofilms

Abstract

Microorganisms have been reported to induce settlement and metamorphosis in a wide range of marine invertebrate species. However, the primary cue reported for metamorphosis of coral larvae is calcareous coralline algae (CCA). Herein we report the community structure of developing coral reef biofilms and the potential role they play in triggering the metamorphosis of a scleractinian coral. Two-week-old biofilms induced metamorphosis in less than 10% of larvae, whereas metamorphosis increased significantly on older biofilms, with a maximum of 41% occurring on 8-week-old microbial films. There was a significant influence of depth in 4- and 8-week biofilms, with greater levels of metamorphosis occurring in response to shallow-water communities. Importantly, larvae were found to settle and metamorphose in response to microbial biofilms lacking CCA from both shallow and deep treatments, indicating that microorganisms not associated with CCA may play a significant role in coral metamorphosis. A polyphasic approach consisting of scanning electron microscopy, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) revealed that coral reef biofilms were comprised of complex bacterial and microalgal communities which were distinct at each depth and time. Principal-component analysis of FISH data showed that the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Cytophaga-Flavobacterium of Bacteroidetes had the largest influence on overall community composition. A low abundance of Archaea was detected in almost all biofilms, providing the first report of Archaea associated with coral reef biofilms. No differences in the relative densities of each subdivision of Proteobacteria were observed between slides that induced larval metamorphosis and those that did not. Comparative cluster analysis of bacterial DGGE patterns also revealed that there were clear age and depth distinctions in biofilm community structure; however, no difference was detected in banding profiles between biofilms which induced larval metamorphosis and those where no metamorphosis occurred. This investigation demonstrates that complex microbial communities can induce coral metamorphosis in the absence of CCA.

FIG. 1.

Percentage of metamorphosis of A. microphthalma larvae on 2-, 4-, and 8-week-old biofilms. Biofilms were developed at site 1 at two depths, 4 m (S) and 12 m (D). Twenty replicate slides were assessed in each treatment (bars = 1 standard error).

FIG. 2.

Scanning electron micrographs of marine biofilms over time and depth (A to D) and on CCA (E and F), with diatoms indicated by arrows. Early stage metamorphosis of coral larvae in response to reef biofilms developed on glass slides in the presence (G) and absence (H) of CCA is shown, with individual corals indicated by arrows.

FIG. 3.

FISH images of marine biofilms with the Cy5-labeled bacterium-specific probe (EUB338), the Cy3-labeled GAM42a (for Gammaproteobacteria), the fluorescein-labeled ALF1b (for Alphaproteobacteria), and the Cy3-labeled Arch915 (for Archaea). Cells which appear magenta are Gammaproteobacteria, cyan cells are Alphaproteobacteria, blue cells are other bacteria, and red cells are Archaea. The scale bar on all images is 100 μm. Average bacterial counts ± standard errors per microscopic field of view (n = 60) with the Cy5-labeled bacterium-specific probe EUB338 were 160 ± 4.4 (2 weeks, shallow), 146 ± 4.0 (2 weeks, deep), 176 ± 4.7 (4 weeks, shallow), 166 ± 4.3 (4 weeks, deep), 185 ± 5.1 (8 weeks, shallow), and 184 ± 5.3 (8 weeks, deep).

FIG. 4.

Quantitative estimates of biofilm community composition using group-specific FISH probes. All densities are expressed as a percentage of total bacterial numbers obtained using dual hybridization reactions with the bacterium-specific probe EUB338. Site 2 data were collected after 8 weeks of biofilm development. Error bars, standard errors.

FIG. 5.

PCA of biofilm community composition at site 1 using FISH counts to describe the community structure of reef biofilms. Abbreviations: 2W S and 2W D, shallow- and deep-water biofilms, respectively, developed over 2 weeks; 4W S and 4W D, shallow- and deep-water biofilms, respectively, developed over 4 weeks; 8W S and 8W D, shallow- and deep-water biofilms, respectively, developed over 8 weeks. Biplots of the response variables were overlaid on the PCA plots as vectors. A, Actinobacteria; B, Planctomycetales; C, Firmicutes; D, Archaea.

FIG. 6.

The large diversity of bacteria (A) and eukarya (B) associated with marine biofilms was detected using DGGE. All treatments were from site 1. Lanes: 4WS and 4WD, shallow- and deep-water biofilms, respectively, developed over 4 weeks; 8WS and 8WD, shallow- and deep-water biofilms, respectively, developed over 8 weeks. *, induced metamorphosis.

FIG. 7.

Cluster analysis of DGGE banding profiles using binary Euclidean distances and between-group linkage. Abbreviations: S, shallow; D, deep; 4W, 4-week biofilms; 8W, 8-week biofilms. *, induced metamorphosis.