The marine fireworm Hermodice carunculata is a winter reservoir and spring-summer vector for the coral-bleaching pathogen Vibrio shiloi

Vibrio shiloi, the causative agent of bleaching of the coral Oculina patagonica in the Mediterranean Sea, is present in all bleached O. patagonica corals in the summer (25-30 degrees C), but can be not detected in the coral during the winter (16-20 degrees C). Furthermore, the pathogen can not survive in O. patagonica at temperatures below 20 degrees C. Using fluorescence in situ hybridization (FISH) with a V. shiloi-specific oligonucleotide probe, we found that the marine fireworm Hermodice caranculata is a winter reservoir for V. shiloi. Worms taken directly from the sea during the winter contained approximately 10(8) V. shiloi per worm by FISH analysis. However, colony-forming units (cfu) revealed only 4.1-18.3 x 10(4) V. shiloi per worm, indicating that approximately 99.9% of them were in the viable-but-not-culturable (VBNC) state. When worms were infected with V. shiloi, most of the bacteria adhered to the worm within 24 h and then penetrated into epidermal cells. By 48 h, less than 10(-4) of the intact V. shiloi in the worm gave rise to colonies, suggesting that they differentiated inside the worm into the VBNC state. When worms infected with V. shiloi were placed in aquaria containing O. patagonica, all of the corals showed small patches of bleached tissue in 7-10 days and total bleaching in 17 days. This is the first report of a reservoir and vector for a coral disease.     

Superoxide Dismutase Is a Virulence Factor Produced by the Coral Bleaching Pathogen <Emphasis Type="Italic">Vibrio shiloi</Emphasis>

Coral bleaching is a disease that threatens coral reefs throughout the world. The disease is correlated with higher-than-normal seawater temperatures. Data have been reported showing that bleaching of the coral Oculina patagonica during the summer in the Mediterranean Sea is the result of an infection with Vibrio shiloi. The summer temperatures induce the expression of virulence factors in the pathogen. We report here that V. shiloi produces an extracellular superoxide dismutase (SOD) at 30°C, but not at 16°C. An SOD⁻ mutant was avirulent. The mutant adhered to corals, penetrated into coral cells, multiplied intracellularly for a short time, and then died. These data support the hypothesis that SOD protects the intracellular V. shiloi from oxidative stress caused by the high concentration of oxygen produced by intracellular zooxanthellae photosynthesis. Received: 3 July 2002 / Accepted: 27 July 2002     

Microbial diseases of corals and global warming

Coral bleaching and other diseases of corals have increased dramatically during the last few decades. As outbreaks of these diseases are highly correlated with increased sea-water temperature, one of the consequences of global warming will probably be mass destruction of coral reefs. The causative agent(s) of a few of these diseases have been reported: bleaching of Oculina patagonica by Vibrio shiloi; black band disease by a microbial consortium; sea-fan disease (aspergillosis) by Aspergillus sydowii; and coral white plague possibly by Sphingomonas sp. In addition, we have recently discovered that Vibrio coralyticus is the aetiological agent for bleaching the coral Pocillopora damicornis in the Red Sea. In the case of coral bleaching by V. shiloi, the major effect of increasing temperature is the expression of virulence genes by the pathogen. At high summer sea-water temperatures, V. shiloi produces an adhesin that allows it to adhere to a beta-galactoside-containing receptor in the coral mucus, penetrate into the coral epidermis, multiply intracellularly, differentiate into a viable-but-not-culturable (VBNC) state and produce toxins that inhibit photosynthesis and lyse the symbiotic zooxanthellae. In black band disease, sulphide is produced at the coral-microbial biofilm interface, which is probably responsible for tissue death. Reports of newly emerging coral diseases and the lack of epidemiological and biochemical information on the known diseases indicate that this will become a fertile area of research in the interface between microbial ecology and infectious disease.     

Differential effects of temperature and starvation on induction of the viable-but-nonculturable state in the coral pathogens Vibrio shiloi and Vibrio tasmaniensis

We compared induction of the viable-but-nonculturable (VBNC) state in two Vibrio spp. isolated from diseased corals by starving the cells and maintaining them in artificial seawater at 4 and 20 degrees C. In Vibrio tasmaniensis, isolated from a gorgonian octocoral growing in cool temperate water (7 to 17 degrees C), the VBNC state was not induced by incubation at 4 degrees C after 157 days. By contrast, Vibrio shiloi, isolated from a coral in warmer water (16 to 30 degrees C), was induced into the VBNC state by incubation at 4 degrees C after 126 days. This result is consistent with reports of low-temperature induction in several Vibrio spp. A large proportion of the V. tasmaniensis population became VBNC after incubation for 157 days at 20 degrees C, and V. shiloi became VBNC after incubation for 126 days at 20 degrees C. Resuscitation of V. shiloi cells from cultures at both temperatures was achieved by nutrient addition, suggesting that starvation plays a major role in inducing the VBNC state. Our results suggest that viable V. shiloi could successfully persist in the VBNC state in seawater for significant periods at the lower temperatures that may be experienced in winter conditions, which may have an effect on the seasonal incidence of coral bleaching. For both species, electron microscopy revealed that prolonged starvation resulted in transformation of the cells from rods to cocci, together with profuse blebbing, production of a polymer-like substance, and increased membrane roughness. V. shiloi cells developed an increased periplasmic space and membrane curling; these features were absent in V. tasmaniensis.     

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

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.     

The coral bleaching Vibrio shiloi Kushmaro et al. 2001 is a later synonym of Vibrio mediterranei Pujalte and Garay 1986

The coral bleaching Vibrio shiloi LMG 19703T was characterized by means of Fluorescent Amplified Fragment Length Polymorphism (FAFLP), DNA-DNA hybridisation, mol% G+C content, fatty acids methyl ester (FAME) analysis and phenotypical tests. Numerical analysis of the FAFLP band patterns indicated that the type strain of V. shiloi in fact belongs to the species V. mediterranei. The type strains of both species shared 77% DNA similarity, as determined by DNA-DNA hybridisation experiments at stringent conditions. Moreover, V. shiloi and V. mediterranei showed almost identical fatty acid composition and phenotypical features. Collectively, the genotypic and phenotypic data presented in this study suggest that V. shiloi Kushmaro et al. 2001 should be considered a later synonym of V. mediterranei Pujalte and Garay 1986. The involvement of V. mediterranei in coral bleaching was unknown until now.     

Genome analysis of the coral bleaching pathogen <Emphasis Type="Italic">Vibrio shiloi</Emphasis>

The past few decades have seen a world-wide increase in coral diseases, yet little is known about coral pathogens. In this study, techniques commonly used in pathogenomic research were applied to the coral pathogen Vibrio shiloi in order to identify genetic elements involved in its virulence. Suppressive subtractive hybridization was used to compare the gene content of V. shiloi to that of a closely related but non-pathogenic bacterium, Vibrio mediterranei, resulting in identification of several putative virulence factors and of three novel genomic islands. The entire genome of V. shiloi was further screened for genes related to previously characterized steps in infection: adhesion, superoxide dismutase production and toxin production. Exposure of pure cultures of V. shiloi to crushed coral tissues strongly affected the expression of seven genes encoding pili, zona occludins toxin (Zot) and a superoxide dismutase. Analysis of eight V. shiloi strains isolated in the last decade shows a shift of the natural population from strains carrying all three genomic islands to strains carrying none of them. This shift occurred following appearance of resistance in the coral Oculina patagonica to infection by V. shiloi. The relevance of these findings to the bleaching disease caused by V. shiloi is discussed.     

Inhibition of photosynthesis and bleaching of zooxanthellae by the coral pathogen Vibrio shiloi

Vibrio shiloi is the causative agent of bleaching (loss of endosymbiotic zooxanthellae) of the coral Oculina patagonica in the Mediterranean Sea. To obtain information on the mechanism of bleaching, we examined the effect of secreted material (AK1-S) produced by V. shiloi on zooxanthellae isolated from corals. AK1-S caused a rapid inhibition of photosynthesis of the algae, as measured with a Mini-PAM fluorometer. The inhibition of photosynthesis was caused by (i) ammonia produced during the growth of V. shiloi on protein-containing media and (ii) a non-dialysable heat-resistant factor. This latter material did not inhibit photosynthesis of the algae by itself but, when added to different concentrations of NH₄Cl, enhanced the inhibition approximately two- to threefold. Ammonia and the enhancer were effective to different degrees on zooxanthellae isolated from four species of coral examined. In addition to the rapid inhibition of photosynthesis, AK1-S caused bleaching (loss of pigmentation) and lysis of zooxanthellae. Bleaching was more rapid than lysis, reaching a peak (25% bleached algae) after 6 h. The factors in AK1-S responsible for bleaching and lysis were different from those responsible for the inhibition of photosynthesis, because they were heat sensitive, non-dialysable and active in the dark. Thus, the coral pathogen V. shiloi produces an array of extracellular materials that can inhibit photosynthesis, bleach and lyse zooxanthellae.     

Temperature-regulated bleaching and lysis of the coral Pocillopora damicornis by the novel pathogen Vibrio coralliilyticus

Coral bleaching is the disruption of symbioses between coral animals and their photosynthetic microalgal endosymbionts (zooxanthellae). It has been suggested that large-scale bleaching episodes are linked to global warming. The data presented here demonstrate that Vibrio coralliilyticus is an etiological agent of bleaching of the coral Pocillopora damicornis. This bacterium was present at high levels in bleached P. damicornis but absent from healthy corals. The bacterium was isolated in pure culture, characterized microbiologically, and shown to cause bleaching when it was inoculated onto healthy corals at 25 degrees C. The pathogen was reisolated from the diseased tissues of the infected corals. The zooxanthella concentration in the bacterium-bleached corals was less than 12% of the zooxanthella concentration in healthy corals. When P. damicornis was infected with V. coralliilyticus at higher temperatures (27 and 29 degrees C), the corals lysed within 2 weeks, indicating that the seawater temperature is a critical environmental parameter in determining the outcome of infection. A large increase in the level of the extracellular protease activity of V. coralliilyticus occurred at the same temperature range (24 to 28 degrees C) as the transition from bleaching to lysis of the corals. We suggest that bleaching of P. damicornis results from an attack on the algae, whereas bacterium-induced lysis and death are promoted by bacterial extracellular proteases. The data presented here support the bacterial hypothesis of coral bleaching.     

Proline-rich peptide from the coral pathogen Vibrio shiloi that inhibits photosynthesis of Zooxanthellae

The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29 degrees C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH(4)Cl, pure natural or synthetic toxin P (10 microM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH(4)Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH(4)Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH(3) into the cell. It is known that uptake of NH(3) into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.     

Neutralization of radical toxicity by temperature-dependent modulation of extracellular SOD activity in coral bleaching pathogen Vibrio shiloi and its role as a virulence factor

Vibrio shiloi is the first and well-documented bacterium which causes coral bleaching, particularly, during summer, when seawater temperature is between 26 and 31°C. Coral bleaching is the disruption of the symbiotic association between coral hosts and their photosynthetic microalgae zooxanthellae. This is either due to lowered resistance in corals to infection or increased virulence of the bacterium at the higher sea surface temperature. The concentration of the oxygen and resulting oxygen radicals produced by the zooxanthellae during photosynthesis are highly toxic to bacteria, which also assist corals in resisting the infection. Hence, in this study we examined the effect of different temperatures on the activity of a novel extracellular SOD in V. shiloi. We also partially characterized the SOD and clearly confirmed that the extracellular SOD produced by V. shiloi is Mn–SOD type, as it was not inhibited by H₂O₂ or KCN. Performing chemical susceptibility killing assay, we confirmed that extracellular SOD may act as first line of defense for the bacteria against the reactive oxygen species. Since, increased activity of novel Mn–SOD at higher temperature, leads to the neutralization of radical toxicity and facilitates the survival of V. shiloi. Hence, the extracellular Mn–SOD may be considered as a virulence factor.     

Relationships among thermal stress, bleaching and oxidative damage in the hermatypic coral, Pocillopora capitata

To examine the response to exposure to a thermal gradient in coral, we assessed the effect of a gradual 10 degrees C temperature increase (22 to 32 degrees C over 10 h) on normal (N), partially bleached (P) and control (C) samples collected from different branches of the same coral (Pocillopora capitata). We examined markers of oxidative stress, including lipid peroxidation (MDA) and superoxide dismutase (SOD) activity, indicators of bleaching, including chlorophyll a (Chl a) and carotenoid pigment (PC) levels, as well as zooxanthellae density. Our results revealed that N, P and C coral samples all contained higher levels of PC versus Chl a. The levels of both pigments increased as the temperature increased from 22 to 28 degrees C only in N and C samples, whereas P samples showed less cellular damage than N and C samples at temperatures between 26 and 28 degrees C, and had greater antioxidant activities at temperatures between 26 and 30 degrees C. The rate of zooxanthellar expulsion consistently increased with temperature in all three coral types across the entire temperature range. Collectively, these results indicate that temperature has a direct effect on the antagonistic relationship between temperature-induced damage and protective antioxidant mechanisms in this type of coral.     

Effect of Temperature on Adhesion of Vibrio Strain AK-1 to Oculina patagonica and on Coral Bleaching

Laboratory aquarium experiments demonstrated that Vibrio strain AK-1 caused rapid and extensive bleaching of the coral Oculina patagonica at 29 degrees C, slower and less-complete bleaching at 23 degrees C, and no bleaching at 16 degrees C. At 29 degrees C, the application of approximately 100 Vibrio strain AK-1 cells directly onto the coral caused 50 and 83% bleaching after 10 and 20 days, respectively. At 16 degrees C, there was no bleaching, even with an initial inoculum of 1.2 x 10 bacteria. To begin to understand the effect of seawater temperature on bleaching of O. patagonica by Vibrio strain AK-1, adhesion of the bacteria to the coral as a function of temperature was studied. Inoculation of 10Vibrio strain AK-1 organisms into flasks containing 20 ml of seawater at 25 degrees C and a fragment of O. patagonica resulted in net levels of bacterial adhesion to the coral of 45, 78, and 84% after 2, 6, and 8 h, respectively. The adhesion was inhibited 65% by 0.001% d-galactose and 94% by 0.001% methyl-beta-d-galactopyranoside (beta-M-Gal). After the incubation of Vibrio strain AK-1 with the coral for 6 h, 42% of the input bacteria were released from the coral with 0.01% beta-M-Gal, compared to less than 0.2% when beta-M-Gal was present during the adhesion step. Adhesion did not occur when Vibrio strain AK-1 was grown at 16 degrees C, regardless of whether the corals were maintained at 16 or 25 degrees C, whereas bacteria grown at 25 degrees C adhered to corals maintained at 16 or 25 degrees C. Bacteria grown at 25 degrees C adhered avidly to Sepharose beads containing covalently bound beta-d-galactopyranoside but failed to bind if grown at 16 degrees C. These data suggest that elevated seawater temperatures may cause coral bleaching by allowing for the expression of adhesin genes of Vibrio strain AK-1.     

Response of Hawaiian and other Indo-Pacific reef corals to elevated temperature

Loss of symbiotic zooxanthellae, or bleaching is one of the first visible signs of thermal stress. Critical threshold temperatures for coral bleaching vary geographically, but can be expressed universally as fixed increments relative to the historical mean local summer maximum. Bleaching can be induced by short-term exposure (i.e. 1–2 days) at temperature elevations of 3°C to 4°C above normal summer ambient or by long-term exposure (i.e. several weeks) at elevations of 1°C to 2°C. Corals in both tropical and subtropical locations live at temperatures close to their lethal limits during the summer months. Temperature elevations above summer ambient, but still below the bleaching threshold, can impair growth and reproduction. Temperature and light interact synergistically; high light accelerates bleaching caused by elevated temperature. Bleaching susceptibility is correlated with respiration rate. Any factor that increases respiration (such as high incident light) accelerates bleaching at higher temperatures. Ultraviolet (UV) radiation is a detrimental factor associated with solar radiation. Increased UV due to thinning of the earth's protective ozone layer may aggravate bleaching and mortality caused by global warming. A warming trend in Hawaiian waters has been observed over the past decade. In 1986, 1987 and 1988 Hawaiian corals were perilously close to their bleaching threshold during the summer months, and localized bleachings did occur. In some cases, local warming of surface water on shallow reef flats exceeded this threshold temperature and caused localized coral bleaching. In other cases, heating of large mesoscale eddies in the lee of the larger islands apparently caused wide-scale bleaching of the most sensitive coral species (Pocillopora meandrina) to depths of 20 m. A continuation of the warming trend in Hawaii would lead to mass bleachings similar to those observed recently in other geographic locations.     

Antimicrobial properties of resident coral mucus bacteria of Oculina patagonica

The inhibitory properties of the microbial community of the coral mucus from the Mediterranean coral Oculina patagonica were examined. Out of 156 different colony morphotypes that were isolated from the coral mucus, nine inhibited the growth of Vibrio shiloi , a species previously shown to be a pathogen of this coral. An isolate identified as Pseudoalteromonas sp. was the strongest inhibitor of V. shiloi . Several isolates, especially one identified as Roseobacter sp., also showed a broad spectrum of action against the coral pathogens Vibrio coralliilyticus and Thallassomonas loyana , plus nine other selected Gram-positive and Gram-negative bacteria. Inoculation of a previously established biofilm of the Roseobacter strain with V. shiloi led to a 5-log reduction in the viable count of the pathogen within 3 h, while inoculation of a Pseudoalteromonas biofilm led to complete loss of viability of V. shiloi after 3 h. These results support the concept of a probiotic effect on microbial communities associated with the coral holobiont.     

Antagonistic interactions among coral-associated bacteria

Reef-building corals are comprised of close associations between the coral animal, symbiotic zooxanthellae, and a diversity of associated microbes (including Bacteria, Archaea and Fungi). Together, these comprise the coral holobiont – a paradigm that emphasizes the potential contributions of each component to the overall function and health of the coral. Little is known about the ecology of the coral-associated microbial community and its hypothesized role in coral health. We explored bacteria–bacteria antagonism among 67 bacterial isolates from the scleractinian coral Montastrea annularis at two temperatures using Burkholder agar diffusion assays. A majority of isolates exhibited inhibitory activity (69.6% of isolates at 25°C, 52.2% at 31°C), with members of the γ-proteobacteria ( Vibrionales and Alteromonadales ) being especially antagonistic. Elevated temperatures generally reduced levels of antagonism, although the effects were complex. Several potential pathogens were observed in the microbial community of apparently healthy corals, and 11.6% of isolates were able to inhibit the growth of the coral pathogen Vibrio shiloi at 25°C. Overall, this study demonstrates that antagonism could be a structuring force in coral-associated microbial communities and may contribute to pathogenesis as well as disease resistance.     

Coral disease following massive bleaching in 2005 causes 60% decline in coral cover on reefs in the US Virgin Islands

In the northeast Caribbean, doldrum-like conditions combined with elevated water temperatures in the summer/fall 2005 created the most severe coral bleaching event ever documented within this region. Video monitoring of 100 randomly chosen, permanent transects at five study sites in the US Virgin Islands revealed over 90% of the scleractinian coral cover showed signs of thermal stress by paling or becoming completely white. Lower water temperatures in October allowed some re-coloring of corals; however, a subsequent unprecedented regional outbreak of coral disease affected all sites. Five known diseases or syndromes were recorded; however, most lesions showed signs similar to white plague. Nineteen scleractinian species were affected by disease, with >90% of the disease-induced lesions occurring on the genus Montastraea. The disease outbreak peaked several months after the onset of bleaching at all sites but did not occur at the same time. The mean number of disease-induced lesions increased 51-fold and the mean area of disease-associated mortality increased 13-fold when compared with pre-bleaching disease levels. In the 12 months following the onset of bleaching, coral cover declined at all sites (average loss: 51.5%, range: 42.4–61.8%) reducing the five-site average from 21.4% before bleaching to 10.3% with most mortality caused by white plague disease, not bleaching. Continued losses through October 2007 reduced the average coral cover of the five sites to 8.3% (average 2-year loss: 61.1%, range: 53.0–79.3%). Mean cover by M. annularis (complex) decreased 51%, Colpophyllia natans 78% and Agaricia agaricites 87%. Isolated disease outbreaks have been documented before in the Virgin Islands, but never as widespread or devastating as the one that occurred after the 2005 Caribbean coral-bleaching event. This study provides insight into the effects of continued seawater warming and subsequent coral bleaching events in the Caribbean and highlights the need to understand links between coral bleaching and disease.     

Sea surface temperatures and coral reef bleaching off La Parguera, Puerto Rico (northeastern Caribbean Sea)

 Much recent attention has been given to coral reef bleaching because of its widespread occurrence, damage to reefs, and possible connection to global change. There is still debate about the relationship between temperature and widespread bleaching. We compared coral reef bleaching at La Parguera, Puerto Rico to a 30-y (1966–1995) record of sea surface temperature (SST) at the same location. The last eight years of the La Parguera SST record have all had greater than average maximum temperatures; over the past 30 y maximum summer temperature has increased 0.7 °C. Coral reef bleaching has been particularly frequent since the middle 1980s. The years 1969, 1987, 1990, and 1995 were especially noteworthy for the severity of bleaching in Puerto Rico. Seven different annual temperature indices were devised to determine the extent to which they could predict severe coral bleaching episodes. Three of these, maximum daily SST, days >29.5 °C, and days >30 °C predict correctly the four years with severe bleaching. A log-log linear relationship was found between SST and the number of days in a given year above that SST at which severe coral beaching was observed. However, the intra-annual relationship between temperature and the incidence of bleaching suggests that no one simple predictor of the onset of coral bleaching within a year may be applicable. Accepted: 17 March 1998     

Temperature-Regulated Bleaching and Lysis of the Coral Pocillopora damicornis by the Novel Pathogen Vibrio coralliilyticus

Coral bleaching is the disruption of symbioses between coral animals and their photosynthetic microalgal endosymbionts (zooxanthellae). It has been suggested that large-scale bleaching episodes are linked to global warming. The data presented here demonstrate that Vibrio coralliilyticus is an etiological agent of bleaching of the coral Pocillopora damicornis. This bacterium was present at high levels in bleached P. damicornis but absent from healthy corals. The bacterium was isolated in pure culture, characterized microbiologically, and shown to cause bleaching when it was inoculated onto healthy corals at 25°C. The pathogen was reisolated from the diseased tissues of the infected corals. The zooxanthella concentration in the bacterium-bleached corals was less than 12% of the zooxanthella concentration in healthy corals. When P. damicornis was infected with V. coralliilyticus at higher temperatures (27 and 29°C), the corals lysed within 2 weeks, indicating that the seawater temperature is a critical environmental parameter in determining the outcome of infection. A large increase in the level of the extracellular protease activity of V. coralliilyticus occurred at the same temperature range (24 to 28°C) as the transition from bleaching to lysis of the corals. We suggest that bleaching of P. damicornis results from an attack on the algae, whereas bacterium-induced lysis and death are promoted by bacterial extracellular proteases. The data presented here support the bacterial hypothesis of coral bleaching.     

Defences against oxidative stress in vibrios associated with corals

Bacteria colonizing healthy coral tissue may produce enzymes capable of overcoming the toxic effects of reactive oxygen species, including superoxide dismutase (SOD) and catalase. Significant differences in the activities of these enzymes were observed in cultures of Vibrio campbellii, Vibrio coralliilyticus, Vibrio harveyi, Vibrio mediterranei, Vibrio pelagius, Vibrio rotiferanus, Vibrio tasmaniensis, and Photobacterium eurosenbergii isolated from healthy, bleached or necrotic tropical and cold water corals. Levels of SOD in exponential phase cultures of V. coralliilyticus grown at 28 degrees C were only slightly higher than those grown at 16 degrees C whereas the levels in stationary phase cultures at 28 degrees C were 7.3 x higher than those at 16 degrees C. The increase in catalase activity of V. coralliilyticus and V. harveyi upon entry to stationary phase conferred protection against killing by oxidative stress. Increased temperature affected up-regulation of enzymes in stationary phase cultures, but pretreatment of cultures with hydrogen peroxide had no significant effect on induction of catalase or SOD. The increased activities appear to be due to up-regulation of gene expression rather than induction of different forms of the enzymes. We suggest that SOD and catalase are unlikely to be major factors in the virulence of these bacteria for corals and that their main function may be to protect against endogenous superoxide.