Specificity of associations between bacteria and the coral Pocillopora meandrina during early development

Abstract

Relationships between corals and specific bacterial associates are thought to play an important role in coral health. In this study, the specificity of bacteria associating with the coral Pocillopora meandrina was investigated by exposing coral embryos to various strains of cultured marine bacteria, sterile seawater, or raw seawater and examining the identity, density, and location of incorporated cells. The isolates utilized in this experiment included members of the Roseobacter and SAR11 clades of the Alphaproteobacteria, a Pseudoalteromonas species of the Gammaproteobacteria, and a Synechococcus species of the Cyanobacteria phylum. Based on terminal restriction fragment length polymorphism analysis of small-subunit rRNA genes, similarities in bacterial communities associated with 170-h-old planulae were observed regardless of treatment, suggesting that bacteria may have been externally associated from the outset of the experiment. Microscopic examination of P. meandrina planulae by fluorescence in situ hybridization with bacterial and Roseobacter clade-specific oligonucleotide probes revealed differences in the densities and locations of planulae-associated cells. Planulae exposed to either raw seawater or strains of Pseudoalteromonas and Roseobacter harbored the highest densities of internally associated cells, of which 20 to 100% belonged to the Roseobacter clade. Planulae exposed to sterile seawater or strains of the SAR11 clade and Synechococcus did not show evidence of prominent bacterial associations. Additional analysis of the raw-seawater-exposed planulae via electron microscopy confirmed the presence of internally associated prokaryotic cells, as well as virus-like particles. These results suggest that the availability of specific microorganisms may be an important factor in the establishment of coral-bacterial relationships.

Fig 1

Heat map depicting the relative abundance of bacterial T-RFs from 170-h-old P. meandrina planulae and the medium treatment water after two different 24-h bacterial culture exposures (72 and 170 h). Percentages in the T-RFLP profiles, shown in the color key, are from combined forward and reverse profiles. Also shown are the T-RFLP profiles for all bacterial culture and seawater treatments at T = 0.

Fig 2

Confocal epifluorescence micrographs of 170-h-old P. meandrina planulae reared in the presence of Roseobacter clade strain HIMB1 (a, b), Pseudoalteromonas strain HIMB1276 (c, d), and raw seawater (e, f). Bacterial cells were observed in planulae hybridized with the bacterial probe suite (indicated by arrows in panels a, c, and e) but not in control samples exposed to a nonsense control probe (b, d, f). Coral tissues and zooxanthellae (representative cells indicated with “z”) are illuminated due to autofluorescence in all images. Gland cells and/or nematocysts (g/n) are prominent in samples due to nonspecific hybridization. The ectodermal (ec) and endodermal (en) differentiation is drawn for reference.

Fig 3

Confocal epifluorescence micrographs of 170-h-old P. meandrina planulae hybridized with a probe specific for cells of the Roseobacter clade. Planulae reared in the presence of Roseobacter clade strain HIMB1 (a) and raw seawater (b) are shown. Cells of the Roseobacter clade are indicated with arrows. The line differentiates the ectodermal (ec) and endodermal (en) tissues.

Fig 4

Transmission electron micrographs of prokaryotic cells and virus-like particles (VLPs) within the ectodermal tissues of 170-h-old P. meandrina planulae. (a to c) Single prokaryotic cells sized 0.4 to 0.7 μm in diameter are located in the ectodermal tissue near the exterior (ex) edge of the planulae. (d) A cluster of VLPs (each VLP sized 40 to 70 nm) near the exterior edge. (e) VLPs near a nucleus (n). (f) Multiple clusters of VLPs near the exterior. Scale bars are 500 nm for panels a to d and 1 μm for panels e and f.