Black band disease (BBD) is a ]]>
Roseofilum reptotaenium has been implicated as the primary pathogen of BBD, but other consortium members may be secondary pathogens that are necessary to the development of the disease. It is known that populations of the sulfate-reducing bacterium Desulfovibrio are present in BBD and that these populations generate sulfide within the band as a byproduct of ]]>
R. reptotaenium. Coral fragments of Montastraea cavernosa and Siderastrea siderea were transferred into two ]]>
R. reptotaenium were inoculated onto the surface of experimental coral fragments. Both M. cavernosa (n = 6) and S. siderea (n=4) became infected and developed BBD-like infections in the control ]]>
M. cavernosa (n=3) or S. siderea (n=2) in the experimental tank containing sodium molybdate. The results of this study reveal that a secondary pathogen is essential to the infection process and ]]>
Desulfovibrio are required for the development of BBD on the coral host. This is the first step in understanding the roles of secondary pathogens in a complex, polymicrobial coral disease.
Key words: Black band disease, sulfate-reducing bacteria, coral.
Resumen
La enfermedad de la Banda Negra ]]>
Roseofilum reptotaenium se considera como el principal patógeno de BBD, pero otros miembros del consorcio pueden ser patógenos secundarios que son ]]>
Desulfovibrio están presentes en BBD y que estas poblaciones generan sulfuro dentro de la banda como un subproducto de la reducción del sulfato. También se conoce que la exposición de los corales sanos a sulfuro conduce a la lisis celular y la muerte del tejido de coral. Trabajos previos muestran que cuando recién ]]>
R. reptotaenium. Fragmentos de coral de Montastraea ]]>
y Siderastrea siderea se transfirieron a dos acuarios experimentales, uno control con solo agua de mar artificial (ASW) y el segundo que contenía ASW y molibdato de sodio 2mM. Pequeñas esteras de cultivo R. ]]>
se inocularon en la superficie de fragmentos experimentales de coral. Tanto M. cavernosa (n=6) y S. siderea (n=4) se ]]>
M. cavernosa (n=3) o S. siderea (n=2) en el tanque experimental con molibdato de sodio. Los resultados de este ]]>
Desulfovibrio son necesarios para el desarrollo de BBD en el coral hospedero. Este es el primer paso en la comprensión de las funciones de los patógenos secundarios en una enfermedad polimicrobiana compleja de coral. ]]>
Palabras clave: Enfermedad de banda, sulfato reductor de bacterias, coral.
Coral diseases were first reported in the 1970s on reefs of the Caribbean (Antonius, 1976; Garret & ]]>
in situ nutrient dosing ]]>
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A wide range of diseases afflict corals on reefs in the wider Caribbean (Weil, 2004). Of these, BBD is of particular interest because it kills large, reef-building coral. BBD is comprised of by a polymicrobial consortium of cyanobacteria, sulfate-reducing and sulfide-oxidizing bacteria, and a diverse assemblage of heterotrophic bacteria that together form a dark, band-shaped microbial mat (Antonius, 1976; Garret & Ducklow, 1975; Ducklow & Mitchell, 1979; Cooney et al., 2002; Frias-Lopez, Zerkle, Bonheyo & Fouke, 2002; Frias-Lopez, Klaus, Bonheyo & Fouke, ]]>
BBD infection can be easily modeled in a laboratory setting due to the highly infectious nature of the disease. In the model system, infection can be initiated when an inoculum of naturally-occurring BBD mat from an infected coral is placed onto a healthy coral fragment (Antonius, 1985). The inoculum can ]]>
There has been a lot of interest in identifying a primary pathogen of BBD. The major target has been BBD cyanobacteria, since the biomass of the band is always dominated by filamentous cyanobacteria (Antonius, 1976). The other members of the consortium –sulfate reducing bacteria (SRB) and sulfide oxidizing bacteria (Ducklow & Mitchell, 1979), as well as heterotrophic ]]>
A critical member of the BBD consortium is Roseofilum reptotaenium (Casamatta, Stanić, Gantar & ]]>
Roseofilum reptotaenium is a gliding, filamentous, phycoerythrin-rich cyanobacterium detected in BBD mats of tropical and sub-tropical coral reefs around the world (Miller & Richardson, 2011). This cyanobacterium forms the matrix of the mat and is adapted to the harsh BBD conditions because of its ability to conduct sulfide-insensitive oxygenic photosynthesis (Myers, Sekar & Richardson, 2007). The ability to conduct photosynthesis in the ]]>
Roseofilum reptotaenium can perform sulfide-insensitive oxygenic photosynthesis in the presence of 0.5mM sulfide (Myers & Richardson, 2009), comparable to sulfide levels measured in intact ]]>
In 2009, samples of BBD were collected from BBD-infected Diploria strigosa and Siderastrea siderea ]]>
R. reptotaenium were isolated from the mat, designated as strain 101-1 (isolated from D. strigosa) and 100-1 (isolated from S. siderea). ]]>
D. strigosa and S. siderea coral fragments to both strains led to BBD-like lesions that ultimately killed the fragments. The inoculum of each strain attached to scleractinian coral tissue, caused an infection, and eventually ]]>
Additional work on BBD pathogenicity has targeted SRB associated with the disease, since the population of SRB is the source of toxic sulfide present in the band (Carlton & Richardson, 1995; Glas et al., 2012). To investigate the role of SRB in BBD pathogenesis, Richardson et al. (2009) used the coral fragment model of BBD infection to assess the effect of sodium ]]>
Montastraea annularis and S. siderea
with freshly collected BBD from infected colonies on the reef. The inoculum consisted of the fully developed consortium of BBD microbes. Coral fragments that were inoculated with BBD that was exposed to 2mM of Na molybdate prior to inoculation did not develop BBD infections (Richardson et al., 2009). Conversely, coral fragments infected with inocula that were not exposed to Na molybdate all developed actively migrating BBD lesions. When these infected fragments were then exposed to 2mM Na molybdate there was no effect on disease progression. These results demonstrated that when the entire BBD community is present, SRB ]]>
The specific role of SRB as potential primary and/or secondary pathogens of BBD infection and development remains unknown. It may be that SRB are acting together with other, as yet unidentified, BBD bacteria to initiate the disease. ]]>
R. reptotaenium for the inoculum. We hypothesized that the biomass of R. reptotaenium creates an anaerobic environment on the coral surface; that this environment ]]>
Materials and Methods
Fragments of two Caribbean ]]>
Montastraea cavernosa and S. siderea, were collected from the Florida Keys National Marine Sanctuary in Key West (Permit number FKMNS-2012-153). After collection, fragments were maintained at ambient (reef water) temperature during transport to ]]>
Two unialgal cultures of R. reptotaenium were used as inocula for the ]]>
M. cavernosa and S. ]]>
respectively. While strain 101-1 was not isolated from BBD on M. cavernosa, preliminary results proved it was infectious for this Caribbean coral species. To inoculate the experimental coral fragments, fragments were taken out of the water and a small amount of R. ]]>
biomass was placed on the fragment surface between polyps. A sterile glass pipette was used to hold the biomass onto the coral surface. Coral fragments were then returned to the aquaria with the pipette in place. Once the biomass attached to the surface of the coral, 24-48 hours later, the pipette was removed. Following pipette removal, if the biomass was sloughed from the surface, a reinfection was attempted in the same area. Reinfections were attempted at least twice on coral fragments with failed inoculations. When infection occurred, progression of the lesion ]]>
The laboratory culture of R. reptotaenium strain 101-1 was photographed using a Leica Application Suite (version 2.8.1) imaging system with a Leica/Leitz DMRB light ]]>
Results
In total, 21 experiments were conducted using 18 coral fragments. Six of eight M. cavernosa fragments and four of five S. siderea fragments from the control (ASW only) aquarium were successfully infected using R. reptotaenium
strain 101-1 (Fig. 1) and 100-1 respectively. In infected coral fragments, the inoculum of R. reptotaenium filaments was observed to attach to the surface of the coral tissue within seven days (Fig. 2). The small amount of inoculum developed into a BBD-like lesion as the ]]>
Fig. 2C-F). Thin tendrils of groups of cyanobacterial filaments could be observed to migrate between and into the coral polyps ahead of the advancing microbial mat (Fig. 2C). Over a period of several weeks, the infection grew into a BBD lesion that migrated across the coral surface (Fig. 2G), lysing tissue (Fig. 2H), and leaving a bare coral skeleton (
Fig. 2I). Similar to naturally occurring BBD, the mat did not tightly attach to the surface of the coral and could be easily disturbed or dislodged from the coral surface.
In contrast to the controls, none of the experimental (ASW plus Na molybdate) fragments –three
M. cavernosa fragments and two S. siderea fragments– became infected (Fig. 3). While the R. reptotaenium inoculum was observed to attach to the ]]>
Fig. 3A), within 72 hours following attachment the filaments were sloughed off of the surface of the coral (Fig. 3B). None of the five fragments exposed to R. reptotaenium in the presence of Na molybdate exhibited permanent attachment to the coral ]]>
R. reptotaenium were placed in the same area on the fragments as previous attempted infections. There were no successful reinfections of the ]]>
Discussion
The results of this study revealed ]]>
R. reptotaenium as the inoculum. Previous work showed that cultured R. reptotaenium can produce BBD in controlled ]]>
in situ. In those experiments the inoculum contained the intact polymicrobial BBD community which always includes active populations of SRB (Miller & Richardson, 2011; Glas et al., 2012).
The two strains of R. reptotaenium used in the experiments presented here are unialgal (each derived from a single cyanobacterial filament), however neither culture is axenic. In each case heterotrophic bacteria are growing in close association with the mucilaginous, polysaccharide-rich, filament sheaths. As is ]]>
R. reptotaenium does not remain viable in an axenic culture for an extended period of time. Stanić (2010) was able to isolate strain 101-1 into axenic culture using the gliding method (Castenholz, 1988), however the culture died within six months. It is generally thought that bacteria associated with the mucilaginous sheath cyanobacteria may provide a necessary vitamin or co-factor for growth and survival. Whatever the underlying ]]>
The bacterial contaminants in the R. reptotaenium cultures were isolated, their 16S rRNA genes sequenced, ]]>
]]>
In our experiments, no SRB were added to the artificial sea water of the control and experimental aquaria. Two potential reservoirs for SRB, however, were the coral fragments and the live-rock used to establish the tank for two weeks prior to the experiment. We theorize that as the cyanobacterial biomass increased at the site of infection on the coral surface, it created an anaerobic environment at the mat-coral interface. The anaerobic environment then enriched for SRB from the reservoirs in the tank, allowing them to proliferate in the anaerobic environment by using ]]>
The presence of high levels of sulfide in BBD has proven to be critical for the pathogenicity of the disease. Using microelectrodes, sulfide levels within BBD have been ]]>
In a series of experiments aimed at studying BBD pathogenesis on the Great Barrier Reef, researchers documented an increase in populations of SRB during a transition of cyanobacterial patches, shown to be precursors of BBD, to fully developed BBD infections (Sato, Willis & Bourne, 2010). In cyanobacterial patches, SRB accounted for less than 1% of the total ]]>
]]>
While different genera of SRB have been identified by sequencing the BBD microbial community, members of the genus Desulfovibrio have been most consistently documented in BBD samples worldwide (Frias-Lopez et al., 2002; Viehman, Mills, Meichel & Richardson, 2006; Sekar et al., 2006; Sekar et al., 2008; Sato et al., 2010). Viehman et al. (2006) cultured and sequenced eight strains ]]>
Desulfovibrio from BBD collected from six coral colonies of four species in the Florida Keys. Desulfovibrio was also detected by Sato et al. (2010) as the SRB in BBD lesions rather than cyanobacterial patches. The presence of Desulfovibrio and other SRB in BBD on different species of coral worldwide strengthens the proposal that SRB are required for all BBD infections.
When comparing our laboratory results using R. ]]>
as the BBD inoculum with the results of Richardson et al. (2009) using the fully developed BBD community, marked differences in infection rate between the cyanobacterial culture and the fully developed BBD inoculum are apparent. While infection of scleractinia coral using naturally occurring BBD occurred within two days, with formation of an actively migrating band within two to three additional days (Richardson et al., 2009), infection using R. reptotaenium took place over a period of weeks. This time lag may be due to the need to recruit SRB from the aquarium environment. These results are in agreement with the results of Sato et al. (2010), who determined that the transition from a cyanobacterial patch to a BBD lesion in situ on the Great Barrier Reef (GBR) occurred over a period of 62±5 days. This time period may similarly be based on the ]]>
R. reptotaenium in the aquarium experiments, or from cyanobacterial patches on the GBR, may be based on the necessity to recruit additional BBD bacteria in addition to the BBD cyanobacterium and SRB.
]]>
The work presented here, together with the body of research to date on the pathogenesis of BBD, strongly suggests that BBD is induced by a sequential community of pathogens, beginning with the cyanobacterium R. reptotaenium, followed by recruitment of SRB, and perhaps involving incorporation of additional BBD bacteria. Based on these findings we propose that SRB are secondary ]]>
Acknowledgments
This work was ]]>
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1. Department of Biological Sciences, Florida International University, Miami, Florida 33199 USA; abrownel@fiu.edu
Received 02-XI-2013 Corrected 02-II-2014 Accepted 24-III-2014