Penetration of the coral-bleaching bacterium Vibrio shiloi into Oculina patagonica

Abstract

Inoculation of the coral-bleaching bacterium Vibrio shiloi into seawater containing its host Oculina patagonica led to adhesion of the bacteria to the coral surface via a beta-D-galactose receptor, followed by penetration of the bacteria into the coral tissue. The internalized V. shiloi cells were observed inside the exodermal layer of the coral by electron microscopy and fluorescence microscopy using specific anti-V. shiloi antibodies to stain the intracellular bacteria. At 29 degrees C, 80% of the bacteria bound to the coral within 8 h. Penetration, measured by the viable count (gentamicin invasion assay) inside the coral tissue, was 5.6, 20.9, and 21.7% of the initial inoculum at 8, 12, and 24 h, respectively. The viable count in the coral tissue decreased to 5.3% at 48 h, and none could be detected at 72 h. Determination of V. shiloi total counts (using the anti-V. shiloi antibodies) in the coral tissue showed results similar to viable counts for the first 12 h of infection. After 12 h, however, the total count more than doubled from 12 to 24 h and continued to rise, reaching a value 6 times that of the initial inoculum at 72 h. Thus, the intracellular V. shiloi organisms were transformed into a form that could multiply inside the coral tissue but did not form colonies on agar medium. Internalization of the bacteria was accompanied by the production of high concentrations of V. shiloi toxin P activity in the coral tissue. Internalization and multiplication of V. shiloi are discussed in terms of the mechanism of bacterial bleaching of corals.

FIG. 1

Electron micrograph of a section of O. patagonica after infection with V. shiloi.

FIG. 2

Section of O. patagonica 24 h after infection with V. shiloi stained with anti-V. shiloi antibodies.

FIG. 3

Multiplication of V. shiloi bacteria in coral tissue. Healthy pieces of coral were inoculated with 1.6 × 10⁵ bacteria per ml and incubated with shaking as described in Table 1, footnote a. At timed intervals, bacterial adhesion (○) to the coral, culturable internal bacteria (■), and total internal bacteria (▴) were measured. Total internal V. shiloi bacteria were quantitated by using specific anti-V. shiloi antibodies. The ordinate indicates the number of cells relative to the inoculum. Error bars, standard errors.

FIG. 4

Viability of V. shiloi in coral tissue. The experiment was performed as described in the legend to Fig. 3. After incubation with the bacteria for 24 h, the washed coral was crushed and examined by fluorescence microscopy for V. shiloi by staining with anti-V. shiloi antibodies (blue) (a) and viable cells (green) by staining with the Live/Dead BacLight Bacterial Viability Kit (b).

FIG. 5

Production and degradation of V. shiloi photosynthesis inhibitor toxin (toxin P) in coral tissue (⧫) compared to internal viable counts of the bacterium (▴). The experiment was performed as described in Table 1, footnote a. Levels of toxin P activity were determined on extracts of coral tissue.