RESULTS
Inoculation of corals with vibrios.
During preliminary testing, all corals inoculated with one YB bacterial isolate at a time showed a slight yellowing appearance similar to YBD. Bacterial samples were recovered from the experimentally infected corals during these testing trials. Pure cultures of the four bacteria unique to YBD, when inoculated (smeared) onto unaffected corals, resulted in signs resembling those of YBD. It is important to note that when all four bacteria together were smeared onto the coral, YB lesions appeared. Only slight paling was evident when bacterial species were inoculated onto the coral singly or in twos or threes. However, when all four of the Vibrio spp. were added together, classic YBD blotch lesions became evident. All corals remained at 20°C for 48 h with no evidence of advancing yellow lesions. Suspected YBD pathogens were inoculated onto healthy corals. These pathogens, which induced YB lesions in the healthy corals, were then reisolated once again and reinoculated onto healthy corals, causing YB lesions as seen in the field. These isolates were sequenced. Sequence analysis indicated that all 10 isolates could be narrowed down (after sequencing) to four isolates that are 99% similar to Vibrio spp.
Inoculation with other bacteria.
E. coli, as well as several other species of bacteria that had been isolated from the corals (including several gram-positive Bacillus spp.), showed no effect on the host tissue or symbiotic algae. No effect on the tissue or symbiotic algae was seen in any of the untreated corals.
Characterization of vibrios.
Two of the four Vibrio strains (YB36 and YBM23) formed cream-colored colonies, while two (YBFL3122 and YBFLG2A) formed yellow colonies. All were gram-negative motile curved rods that grew well at salt concentrations ranging from 1 to 10%. All strains grew on a wide variety of carbon sources. All four Vibrio spp. tested positive for utilization of the following carbon sources: D-mannose, D-galactose, D-lactose, L-fructose, D-fructose, D-mannitol, maltose, m-inositol, D-trehalose, DL-serine, L-leucine, D-gluconic acid, propionic acid, DL-lactic acid, glycerol, DL-[propto]-glycerol-phosphate, and glucose-6-phosphate.
Coupled effects of YBD-infected corals and variation in temperature.
Microscopic observations of all corals exposed to the four Vibrio spp. showed deleterious effects on zooxanthellae at higher temperatures within the gastroderm, leaving the coral host tissue intact at 32 to 33°C. Abnormalities associated with higher temperature and vibrios together were noted. For example, many zooxanthellae were degenerated and fragmented. There is no histological evidence of bacterial penetration into the zooxanthellae.
The mortality of healthy (control) corals exposed to temperatures of 20 to 32°C over time was evident (Fig. 3a). All healthy corals survived the experiments, except those exposed to 32 to 33°C, which experienced 20% mortality after 96 h. No mortality of the remaining controls was evident at any exposure durations. However, closer to the thermal-bleaching threshold, classic signs of temperature-induced expulsion of zooxanthellae were evident.
Mortality of YB-infected corals exposed to different temperatures could also be seen (Fig. 3b). The mortality of YBD-infected corals was highest (60 to 80%) with exposure to 31 to 32°C for 48 and 96 h. The corals experienced 40% mortality at 25°C and 0% mortality at 20°C. It is important to note that no expulsion of zooxanthellae from the yellow lesions was evident.
The rate-of-spread experiments also showed positive results when temperatures were increased. Temperatures were raised to 25°C at 12 h. At hour 24, the temperature was again raised to 30°C, and it was raised to 33°C at 48 and 96 h. The corals were spaced so that none of the specimens were in contact with each other for the duration of the experiment. None of the controls developed YB lesions. Corals with YB lesions that were placed in aquaria and kept at 20°C for 120 h showed no advancement of the yellow lesions at this temperature. The edge of the lesion spread with increasing temperature treatments showed an exponential increase in the yellow lesion (y = –0.0578x2 + 0.7224 x 2; R2 = 0.9931). At the starting point, the average yellow lesion diameter increased in 96 h from 0.74 cm with a temperature treatment of 20°C to 2.2 cm at 33°C toward the end of the experiment. None of the controls developed YB lesions (Fig. 4).
Microscopy, cell densities, mitotic index, and pigment analysis.
Microscopic images of experimentally infected host tissues were similar to those from field specimens of YBD. Corals exposed to YBD at 25°C for 72 h began to show cellular breakdown and necrosis. In YBD corals exposed to 20°C, this breakdown occurred more rapidly (after 48 h) (Fig. 5a and b) than in controls (Fig. 5d). Healthy zooxanthellae were seen surrounded by a bacterial mat 8 h after being inoculated with the four Vibrio spp. (Fig. 5c). No bacteria were evident in symbiotic zooxanthellae, as they were found only in host tissue surrounding the algal cell. Electron micrographs showed redistribution and degeneration of algal organelles (Fig. 6). Vacuolization, fragmentation, swelling, and displacement of cytological features were evident. A 50% decrease in algal density and an 80% decrease in the mitotic index occurred in YBD specimens. Compared with healthy tissue, diseased samples had a 75% decreased concentration of chlorophyll (Chl) a and c2 accessory pigments (Fig. 7 and 8).
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