For microbial analysis, eight 10-ml sterile-syringe samples were drawn from the surface layers of YBD-infected and healthy Montastraea spp., two from the Florida Keys and six from Bonaire, Netherlands Antilles, by scuba diving. Syringe samples were immediately inoculated onto glycerol artificial seawater (GASW) plates (61) and thiosulfate-citrate-bile salt agar plates and then were incubated at 26°C. Colony morphology was examined using a standard stereomicroscope. Bacteria were isolated from the plates, with choices based on distinct morphological and growth characteristics, such as color and shape, and then were subcultured to pure cultures either on GASW or thiosulfate-citrate-bile salt agar.
In total, 143 pure cultures were isolated, 35 originating from control samples (the healthy samples) and 79 originating from diseased coral samples throughout the locations mentioned above. A 48-h agar culture of each isolate was then subjected to carbon source utilization pattern analysis using GN1 96-well microplates (BIOLOG Inc., Hayward, Calif.).
Bacteria associated with YBD were compared with those from healthy Montastraea annularis tissue, based on similarity of carbon source utilization patterns. Sixteen bacterial isolates were chosen, 6 isolates from healthy corals (controls) and 10 isolates that were present only in diseased corals. During preliminary testing, the 10 bacterial isolates from diseased corals were inoculated onto healthy coral tissue in different combinations, and all inoculations resulted in tissue paling. The six bacterial isolates from healthy corals were also inoculated onto healthy coral specimens and induced no yellow lesions.
All 10 isolates that were suspected pathogens were then identified via sequence analysis of the PCR-amplified 16S rRNA gene. DNA isolation from the 10 bacterial isolates was followed by PCR amplification of the 16S rRNA gene, using universal bacterial 16S rRNA gene primers (27F [5'-AGAGTTTGATCCTGGCTAG-3'] and 1522R [5' AAGGAGGTGATCCARCCGCA-3']) (37), and sequencing. Sequencing was performed at the Georgia Tech core DNA facility using a BigDye Terminator version 3.1 cycle-sequencing kit on an automated capillary sequencer (model 3100 Gene Analyzer; Applied Biosystems). All 16S ribosomal DNA sequences were then determined by BLAST analysis from the National Center for Biotechnology Information nucleotide database. The dendrogram displaying phylogenetic positions of the four Vibrio spp. isolated and used in this study was based on alignment of a 511-bp region within the 16S rRNA gene. Sequence alignments were performed using DNA Star Lasergene MEGALIGN software (version 5.06) by the CLUSTAL W method. The tree was generated by the neighbor-joining method (57) using MEGA2 software (version 2.1). Sequence dissimilarity was determined following specific reference points (41). Bootstrap values were based on 500 replicates. Reference strain sequences were obtained from the Ribosomal Database Project (10).
Infection experiments.
Forty healthy coral specimens, each measuring 5 cm square, were used for inoculation experiments. Specimens were collected from Bonaire, Netherlands Antilles, and experiments were carried out at the University of South Carolina and the Mote Marine Laboratory, Summerland Key, Fla.
All corals were placed in outdoor aquarium tanks with fresh running seawater for testing and observation. Ten specimens were not inoculated with any bacteria (controls). Another 10 specimens were inoculated with Escherichia coli bacteria (K-12 strain MG1655; Carolina Biological). Another 10 specimens were inoculated with a mixture of the 10 bacterial strains isolated from the yellow lesion (YBFL3121, YBFL3122, YBM23, YBM22, YB36, YB2F2, YBFLG2A, YB15, YBFL362B, and YB23).
To create the mixture, one pure colony of each of the 10 YBD bacterial strains was cut from the culture plate media and smeared directly onto the surface of each coral. In total, 0.5 ml of mixed bacteria (50 µl from each) was smeared onto each of the 10 corals. The final 10 coral specimens were smeared with 0.5 ml of only 1 of the 10 YBD isolates each, to see if any of the isolates alone would induce the yellow band lesion. The experiment continued for 1 week. The first three groups of 10 were kept in separate tanks, all at 26 to 27.5°C. The corals in the fourth group were kept in 10 separate 1-liter beakers to avoid cross-contamination.
After a week, it became clear that the 10-strain mixture was the only bacterial inoculation causing yellow lesions similar to those found in the field. At that point, a swab from the yellow lesion was reisolated on GASW medium, incubated, and sequenced. Sequencing showed that four distinct strains of Vibrio bacteria were present in the yellow lesion that had been reisolated. (Most of the original 10 bacterial strains were actually duplicate strains of the same four bacteria.) The earlier experiment was then repeated. A combination of these four strains (125 µl each, totaling 500 µl) was smeared onto three different areas on the surface of a single healthy coral.
Cell densities, chlorophyll pigment analysis, and mitotic indices.
Samples were collected from healthy, stressed, and dying regions of Montastraea sp. corals. Total numbers of zooxanthellae and percent mitotic indices (MI) were recorded. The percent MI, which measures the number of dividing cells in a known population of algal cells over time, is calculated by determining the number of dividing algal cells divided by the total number of cells and multiplied by 100, as described by Wilkerson et al. (68).
Coral tissue was collected using a Water Pik (36) and was kept cold in ice-filled coolers. The slurry containing tissue and zooxanthellae was homogenized and centrifuged in a Beckman Accuspin at 5,000 rpm for 5 min to separate the host tissue from zooxanthellae. The liquid was discarded, and the pellet was resuspended in fresh filtered seawater-10% glutaraldehyde solution and recentrifuged for counting. The number of zooxanthellae and percent MI were determined by direct examination under a phase-contrast microscope at x400 and x1,000 magnifications, using a Neubauer ruling hemocytometer. Tissue was also extracted for pigment analysis of chlorophyll a and c2 (35).
Temperature experiments.
Fifteen samples of YBD-infected Montastraea sp. corals and 15 healthy samples from the same colony of Montastraea spp. were collected. The samples were placed in 100-ml polyethylene bottles with fresh filtered seawater and after 1 h were brought to the Mote Marine Laboratory facility. The corals were placed at 23°C and monitored for 48 h to make sure they remained alive for temperature experiments. For the experiments, the corals were placed in outdoor 10-gallon aquarium tanks at three different experimental temperatures: 20, 25, and 32°C. Controls (no YB lesions) were kept in separate tanks at the same temperatures in fresh running seawater. Five corals at 20°C, five at 25°C, and five at 32°C were placed in each experimental tank next to the tanks set up for the controls. This experiment was conducted to investigate the effect of temperature on healthy corals without YB lesions.
Ten 5-cm-square coral samples were collected from a colony with active YB lesions (0.741-cm-diameter average lesion/5-cm square) and placed in 20-gallon aquaria. An additional 10 5-cm-square samples with no yellow lesions on them (controls) were collected and placed in aquaria adjacent to the corals with yellow lesions. All 20 corals were exposed to the same temperatures over time: 20, 25, 30, and 33°C. Three 10-cm-square corals with active YB lesions on them were placed in aquaria and kept between 20 and 33°C for 120 h (the entire experiment). These time intervals were chosen to see how fast the YB lesions could increase during abrupt thermal-bleaching events. These corals were also used as experimental corals for isolation of mucus during bacterial-growth experiments and for monitoring because they had active YB lesions.
Histology and microscopy.
Samples of intact coral tissue displaying macroscopic features of YBD were placed in immersion, fixed in glutaraldehyde, and postfixed in 1.0% osmium tetroxide in phosphate buffer. These tissues were processed for examination by both optical and electron microscopy. After the coral tissues were decalcified, dehydrated in graded alcohols, and embedded in Spurr's epoxy resin, they were sectioned on an LKB Ultratome with a diamond knife. For light microscopy, thick sections were cut at 1.0 to 2.0 µm; for electron microscopy, thin sections were cut at 70 to 90 nm. All sections were prepared using a Dupont diamond knife. Thick sections (
2.0 µm) were mounted on glass microscope slides and stained with aqueous toluidine blue 10B in sodium borate. Thin sections (90 nm) were mounted on Formvar-coated grids and stained with uranyl acetate and lead citrate. Optical photomicroscopy was conducted using a BandL Balplan microscope. Photographs were taken through the 100x objective lens with a film magnification of x300. Fuji 200 ASA color print film was used for all photographic exposures. Electron photomicrographs were recorded on black-and-white film (Kodak 4489) at original magnifications of x8,000 to 34,000.
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