In many diverse ecosystems, ranging from natural surfaces in aquatic ecosystems to the mammalian gut and medical implants, bacterial populations and communities exist as biofilms. While the process of biofilm development has been well-studied for those produced by unicellular bacteria such Pseudomonas aeruginosa, little ]]>
Montastraea annularis, five artificially ]]>
Key words: black band disease, biofilm, microbial mat, corals.
Resumen
En muchos ecosistemas diversos, que van desde ecosistemas acuáticos hasta los intestinos de mamíferos e implantes médicos, las poblaciones y comunidades de bacterias existen como biopelículas (biofilms). El proceso de desarrollo de las biopelículas ha sido bien estudiado para aquellos producidos por bacterias unicelulares como ]]>
Pseudomonas aeruginosa, pero se conoce muy poco acerca del desarrollo de biopelículas asociadas con microorganismos filamentosos. La Enfermedad de Banda Negra (EBN) de coral es caracterizada como una comunidad polimicrobiana que forma una biopelícula (lecho), visualmente-dominada por una cianobacteria filamentosa. El lecho migra a través de un huésped de coral vivo, rompiendo completamente el tejido del coral y dejando atrás el esqueleto de coral expuesto. Es la única biopelícula cianobacteriana que migra a través de un sustrato, por lo tanto ]]>
Montastraea annularis, cinco artificialmente infectados con EBN y dos colectados de una colonia EBN-infectada, fueron usados para abordar estas preguntas mediante exámenes detallados con microscopía electrónica de barrido y de transmisión (MEB y MET). En zonas cercanas a la interfaz de tejido del coral y la banda de la enfermedad madura, se han observado dos tipos de grupos de cianobacterias, uno con orientación ]]>
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Palabras clave: enfermedad banda negra, biopelícula, lecho microbiano, corales Black band disease (BBD) of corals exists as a thick biofilm, or thin mat, that migrates across coral ]]>
et al. 1983). While the mechanisms that control band migration and development are not known, this horizontal migration of the intact biofilm/mat community, and it’s migration across a living coral host, are unique (Richardson 1996). The pathogenicity of a polymicrobial mat dominated by filamentous cyanobacteria is also unique and is an important aspect of the disease.
Biofilms have been ]]>
et al. 2003). Such EPS secretion, which is a conspicuous component of BBD, may play an important role in forming the complex polymicrobial structure associated with this disease. It is well-documented that, when associated in biofims, bacteria exhibit an increased resistance to fluctuating environmental conditions, including avoidance of antibiotics and antagonistic cells associated with host immune systems (Govan and Deretic 1996, Costerton et al. 1999, O’Toole et al. ]]>
Perhaps the most well-studied of pathogenic biofilms are those associated with lung infections in individuals with cystic fibrosis, caused by the unicellular species ]]>
Pseudomonas aeruginosa (O’Toole et al. 2000). Polymicrobial biofilm development has also been studied, for example focusing on biofilms associated with dental plaque, a system that may contain as many as 300 different species of microorganisms (Paster et al. 2001). Model systems have been used to directly examine the process of biofilm development in the laboratory. Such model systems have made use of both single and multiple species, including Escherichia ]]>
, and Vibrio cholerae, among others (Pratt and Kolter 1998, Watnick et al. 1999). However, all of these model systems have focused on unicellular bacteria, and little is known about the development of biofilms associated with filamentous bacteria.
BBD is known to ]]>
et al. 1983, Edmunds 1991, Kuta and Richardson 1996, Sutherland et al. 2004, Voss and Richardson 2006). It is caused by a polymicrobial biofilm, or very thin (<1mm) mat, which can range from a few millimeters to several centimeters in width (Rützler and ]]>
Roseofilum (Casamata et al., 2012) and may also contain members of the cyanobacterial genera Oscillatoria, Geitlerinema, Leptolyngbya and Phormidium (Cooney et al. 2002, Frias-Lopez
et al. 2003, 2004, Sussman et al. 2006, Barneah et al. 2007, Myers et al. 2007). Together, BBD cyanobacteria provide the structural framework for the mat and have recently been shown to be directly involved in BBD pathobiology. BBD-associated coral mortality occurs as the contiguous band migrates across the coral colony, at a rate of three millimeters to one centimeter a day, lysing coral tissue and leaving behind exposed coral skeleton. This coral tissue lysis is aided by a cyanotoxin, ]]>
et al., 2007, 2009; Miller and Richardson 2011). Here we use SEM and TEM to examine the fine structure of the BBD biofilm/mat to elucidate mechanisms that may contribute to the development and progression of the BBD mat across a coral colony.
Examination of the BBD biofilm/mat on infected fragments using SEM revealed that, despite the homogenous appearance of the disease band macroscopically, the mat exhibited spatial heterogeneity. In both artificially and naturally infected coral fragments cyanobacterial filaments were found millimeters ahead ]]>
Figure 1). These filaments were present as loose aggregations that formed clusters between and underneath coral tissue layers, and could be seen separating the coral tissue from the coral skeleton. Some of the clusters (Figure 1A) consisted of cyanobacteria that appeared to be randomly oriented relative to each other, with few filaments in alignment, and had no associated EPS. Other clusters were observed to exhibit active EPS secretion that was associated with ]]>
Figure 1B). In some cases, such filaments appeared to be enveloped in EPS, but there was no distinct layer of EPS matrix holding the filaments together.
The differentiation between EPS and non- EPS producing BBD cyanobacteria can be clearly seen in TEM ]]>
Figure 2). Some clusters of cyanobacteria penetrating through coral tissue had no ring of EPS surrounding each cyanobacterial filament (shown in cross-section in Figure 2A), while other cyanobacteria, also present in clusters and penetrating through coral tissue, had no apparent ring of EPS (Figure 2B).
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Examination of the BBD in the center of the mat (between the leading edge and the exposed coral skeleton behind the band) revealed a much more organized band structure (Figure 3). Thick layers of cyanobacteria were observed to be oriented in parallel and were in much closer physical association than those in the coral tissue in front of the band, providing a distinct structural framework (Figure 3A). It was also observed that parallel filaments ]]>
Figure 3B).
Discussion
In this study, SEM and TEM ]]>
Figure 2), and underneath (Figure 1A) coral tissue. Previous studies have shown that BBD cyanobacteria are able to penetrate into coral tissue (Rützler et al. 1983, Barneah et al. 2007, Sato et al. 2009, Miller
et al. 2011), and into coral skeleton (Miller et al. 2011).
The BBD cyanobacteria ahead of the mature band in the current study were aggregated in clusters that exhibited both primarily random (Figure 1A), or primarily ]]>
Figure 1B), orientation. In these areas of the infected coral the BBD cyanobacteria either exhibited no EPS secretion (Figure 1A, 2A), or were associated with EPS on the surface of filaments (Figure 1B, 2B). Some of these clusters were fully embedded in EPS (
Figure 3B).
There appeared to be a transition between the clusters of cyanobacteria in the tissue in front of the band, and the fully formed band itself. Specifically, randomly oriented, non- EPS forming clusters appeared to transition into parallel-oriented filaments that produced EPS, which then appeared to transition into more closely packed, layered clusters. In some of the ]]>
Figure 3A), which could provide a distinct structural framework for the growth of associated microorganisms in the BBD mat. These aggregations can be considered to be biofilms, which may further aggregate to form the mature mat.
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Previous studies have suggested that BBD cyanobacteria, which are the dominant component of the BBD community, provide the structural framework of the BBD mat (Rützler and Santavy 1983). This hypothesis is supported by the results presented in the current study. Furthermore, the observed layering of filaments, and differentiation in EPS production, are consistent with the results of other studies focused on formation of biofilms. In well-studied, model biofilm systems composed of unicellular bacteria, the development of the biofilm occurs after ]]>
et al. 1999, Flemming et al. 2000, O’Toole et al. 2000, Handley et al. 2001, Rickard et al. 2003). Mutations in the genes controlling EPS secretion lead to both altered attachment behavior and biofilm formation, further ]]>
et al. 1999).
In contrast to the temporal basis of unicellular biofilm formation, it appears that filamentous BBD cyanobacteria form the biofilm/mat in a spatial, longitudinal fashion. As BBD cyanobacteria migrate into tissue ahead of the band, they appear to stop producing EPS. The clusters of filaments behind these leading ]]>
Our results reveal new insights into the development of the BBD biofilm/mat and its association with ]]>
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*Correspondencia: Aaron W. Miller: Department of Biological Sciences, Florida International University, Miami, Florida 33199 USA. aaron.miller@fiu.edu ]]>
Patricia Blackwelder: University of Miami Center for Advanced Microscopy (UMCAM), Miami, Florida 33124, USA. Marine Geology and Geophysics, University of Miami, Miami, Florida 33149,USA. Marine Geology and Geophysics, University of Miami, Miami, Florida 33149,USA. pblackwelder@rsmas.miami.edu Husain Al-Sayegh: University of Miami Center for Advanced Microscopy (UMCAM), Miami, Florida 33124, USA. Marine Geology and Geophysics, University of Miami, Miami, Florida 33149,USA. h.alsayegh@miami.edu Laurie L. Richardson: Department of Biological Sciences, Florida International University, Miami, Florida 33199 USA.richardl@fiu.edu
1. Department of Biological Sciences, Florida International University, Miami, Florida 33199 USA 2. University of Miami Center for Advanced Microscopy (UMCAM), Miami, Florida 33124, USA 3. Marine Geology and Geophysics, University of Miami, Miami, Florida 33149,USA 4. Nova Southeastern University Oceanographic Center, Dania, Florida 33004,USA Corresponding author: email addresses: aaron.miller@fiu.edu, richardl@fiu.edu, pblackwelder@rsmas.miami.edu, h.alsayegh@miami.edu
Received 15-VII-2011. Corrected 12-XII-2011. Accepted 20-XII-2011.