Visualization of coral host--pathogen interactions using a stable GFP-labeled Vibrio coralliilyticus strain

The bacterium Vibrio coralliilyticus has been implicated as the causative agent of coral tissue loss diseases (collectively known as white syndromes) at sites across the Indo-Pacific and represents an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease. In this study, we used a mini-Tn7 transposon delivery system to chromosomally label a strain of V. coralliilyticus isolated from a white syndrome disease lesion with a green fluorescent protein gene (GFP). We then tested the utility of this modified strain as a research tool for studies of coral host–pathogen interactions. A suite of biochemical assays and experimental infection trials in a range of model organisms confirmed that insertion of the GFP gene did not interfere with the labeled strain’s virulence. Using epifluorescence video microscopy, the GFP-labeled strain could be reliably distinguished from non-labeled bacteria present in the coral holobiont, and the pathogen’s interactions with the coral host could be visualized in real time. This study demonstrates that chromosomal GFP labeling is a useful technique for visualization and tracking of coral pathogens and provides a novel tool to investigate the role of V. coralliilyticus in coral disease pathogenesis.Human Frontier Science Program (Strasbourg, France) (No. RGY0089RS

Coral Bleaching Independent of Photosynthetic Activity

SummaryThe global decline of reef-building corals is due in part to the loss of algal symbionts, or “bleaching,” during the increasingly frequent periods of high seawater temperatures [1, 2]. During bleaching, endosymbiotic dinoflagellate algae (Symbiodinium spp.) either are lost from the animal tissue or lose their photosynthetic pigments, resulting in host mortality if the Symbiodinium populations fail to recover [3]. The >1,000 studies of the causes of heat-induced bleaching have focused overwhelmingly on the consequences of damage to algal photosynthetic processes [4–6], and the prevailing model for bleaching invokes a light-dependent generation of toxic reactive oxygen species (ROS) by heat-damaged chloroplasts as the primary trigger [6–8]. However, the precise mechanisms of bleaching remain unknown, and there is evidence for involvement of multiple cellular processes [9, 10]. In this study, we asked the simple question of whether bleaching can be triggered by heat in the dark, in the absence of photosynthetically derived ROS. We used both the sea anemone model system Aiptasia [11, 12] and several species of reef-building corals to demonstrate that symbiont loss can occur rapidly during heat stress in complete darkness. Furthermore, we observed damage to the photosynthetic apparatus under these conditions in both Aiptasia endosymbionts and cultured Symbiodinium. These results do not directly contradict the view that light-stimulated ROS production is important in bleaching, but they do show that there must be another pathway leading to bleaching. Elucidation of this pathway should help to clarify bleaching mechanisms under the more usual conditions of heat stress in the light

Utilization of Mucus from the Coral Acropora palmata by the Pathogen Serratia marcescens and by Environmental and Coral Commensal Bacteria▿ †

In recent years, diseases of corals caused by opportunistic pathogens have become widespread. How opportunistic pathogens establish on coral surfaces, interact with native microbiota, and cause disease is not yet clear. This study compared the utilization of coral mucus by coral-associated commensal bacteria (“Photobacterium mandapamensis” and Halomonas meridiana) and by opportunistic Serratia marcescens pathogens. S. marcescens PDL100 (a pathogen associated with white pox disease of Acroporid corals) grew to higher population densities on components of mucus from the host coral. In an in vitro coculture on mucus from Acropora palmata, S. marcescens PDL100 isolates outgrew coral isolates. The white pox pathogen did not differ from other bacteria in growth on mucus from a nonhost coral, Montastraea faveolata. The ability of S. marcescens to cause disease in acroporid corals may be due, at least in part, to the ability of strain PDL100 to build to higher population numbers within the mucus surface layer of its acroporid host. During growth on mucus from A. palmata, similar glycosidase activities were present in coral commensal bacteria, in S. marcescens PDL100, and in environmental and human isolates of S. marcescens. The temporal regulation of these activities during growth on mucus, however, was distinct in the isolates. During early stages of growth on mucus, enzymatic activities in S. marcescens PDL100 were most similar to those in coral commensals. After overnight incubation on mucus, enzymatic activities in a white pox pathogen were most similar to those in pathogenic Serratia strains isolated from human mucosal surfaces

Coral bleaching independent of photosynthetic activity

The global decline of reef-building corals is due in part to the loss of algal symbionts, or "bleaching," during the increasingly frequent periods of high seawater temperatures [1, 2]. During bleaching, endosymbiotic dinoflagellate algae (Symbiodinium spp.) either are lost from the animal tissue or lose their photosynthetic pigments, resulting in host mortality if the Symbiodinium populations fail to recover [3]. The >1,000 studies of the causes of heat-induced bleaching have focused overwhelmingly on the consequences of damage to algal photosynthetic processes [4-6], and the prevailing model for bleaching invokes a light-dependent generation of toxic reactive oxygen species (ROS) by heat-damaged chloroplasts as the primary trigger [6-8]. However, the precise mechanisms of bleaching remain unknown, and there is evidence for involvement of multiple cellular processes [9, 10]. In this study, we asked the simple question of whether bleaching can be triggered by heat in the dark, in the absence of photosynthetically derived ROS. We used both the sea anemone model system Aiptasia [11, 12] and several species of reef-building corals to demonstrate that symbiont loss can occur rapidly during heat stress in complete darkness. Furthermore, we observed damage to the photosynthetic apparatus under these conditions in both Aiptasia endosymbionts and cultured Symbiodinium. These results do not directly contradict the view that light-stimulated ROS production is important in bleaching, but they do show that there must be another pathway leading to bleaching. Elucidation of this pathway should help to clarify bleaching mechanisms under the more usual conditions of heat stress in the light

Visualization of coral host-pathogen interactions using a stable GFP-labeled Vibrio coralliilyticus strain

The bacterium Vibrio coralliilyticus has been implicated as the causative agent of coral tissue loss diseases (collectively known as white syndromes) at sites across the Indo-Pacific and represents an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease. In this study, we used a mini-Tn7 transposon delivery system to chromosomally label a strain of V. coralliilyticus isolated from a white syndrome disease lesion with a green fluorescent protein gene (GFP). We then tested the utility of this modified strain as a research tool for studies of coral host–pathogen interactions. A suite of biochemical assays and experimental infection trials in a range of model organisms confirmed that insertion of the GFP gene did not interfere with the labeled strain's virulence. Using epifluorescence video microscopy, the GFP-labeled strain could be reliably distinguished from non-labeled bacteria present in the coral holobiont, and the pathogen's interactions with the coral host could be visualized in real time. This study demonstrates that chromosomal GFP labeling is a useful technique for visualization and tracking of coral pathogens and provides a novel tool to investigate the role of V. coralliilyticus in coral disease pathogenesis

Summary of the properties of the counting methods.

<p>^aAt the time of counting. Note that in our standard procedure, the sample is homogenized in 500 μl and then diluted (if needed) to the optimal concentration for counting. This dilution step is not required except for samples to be counted with the Coulter Counter, for which a dilution of ~20-fold into Isoton or some comparable solution is needed. Thus, for counting with the Coulter Counter, the algal concentrations in the original homogenate must be correspondingly higher.</p><p>Summary of the properties of the counting methods.</p

Reliability of total-protein determinations as a measure of total anemone tissue mass.

<p>Symbiotic anemones of strain H2 were homogenized and total protein was measured using our standard protocols (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135725#sec002" target="_blank">Materials and Methods</a>). (a) Comparison of the BCA and DC SDS-compatible protein assays. The total protein in each of four separate homogenates was determined with each assay. Means ± SEMs (n = 3) are shown. (b) Precision of the BCA assay. For each of four separate homogenates, total protein was measured repeatedly with the BCA assay. Means ± SEMs (n = 6) are shown. (c) Correlation between oral-disk diameter and total protein. Twelve anemones of various sizes were anesthetized (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135725#sec002" target="_blank">Materials and Methods</a>) and then photographed using bright-field illumination on the stereomicroscope to measure their oral-disk diameters. Each anemone was then homogenized and its total protein determined in triplicate using the BCA assay. Means ± SEMs are shown. R² = 0.7765 for the correlation shown.</p

Quantification of algal cells using the Guava flow cytometer.

<p>The instrument was operated as described in Materials and Methods; all anemones were of strain CC7. (a) Effects of sample type and preparation method on the flow-cytometer plots of the red (chlorophyll) fluorescence vs. the side-scatter of the particles. The square within each plot indicates the region containing <i>Symbiodinium</i> cells with approximately normal chlorophyll concentration and light scatter. The samples of cultured algae (strain SSA02), fixed anemone homogenate, and coral homogenate (panels 1, 5, and 6) were prepared as described in Materials and Methods. The homogenates of aposymbiotic (APO) and symbiotic (SYM) anemones (panels 2 and 3) were prepared using our standard protocol except (i) the anemones had not been frozen and (ii) the symbiotic anemone was initially homogenized by rotor stator in ASW, after which one-fifth volume of 0.1% SDS in ASW was added before needle shearing and further dilution in 0.1% SDS in dH₂O. The frozen anemones (panel 4) were frozen in 0.1% SDS in ASW, then thawed, homogenized by rotor stator and needle shearing, and diluted further in the same solution. (b and c) Precision of the method and its ability to detect small differences in algal-cell concentrations. Anemone homogenates were prepared and diluted using our standard protocol. (b) Undiluted and diluted homogenates were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135725#pone.0135725.g003" target="_blank">Fig 3g</a> except that n = 8 in this case. (c) Serial two-fold dilutions of the homogenate were prepared, and the predicted (solid line) and measured (as mean values ± SEMs; n = 3) counts were compared. Most error bars are hidden by the points themselves with the graphing program used here. (d) Efficiency of automatic mixing by the instrument. Anemone homogenate was prepared by our standard protocol except that 0.1% SDS in ASW was used for homogenization and dilution. Samples were added to all wells of a 96-well plate, which was then analyzed in the usual way, so that individual samples sat for up to 84 min without agitation except for the automatic mixing that preceded the sampling from each well. R2 for the regression shown = 0.04. (e) Quantification of algal cells in two coral nubbins that had been held at 26°C and in two nubbins that had been stressed at 33°C for 3 d (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135725#sec002" target="_blank">Materials and Methods</a>). Homogenates were prepared as described in Materials and Methods, and algal cell numbers (see a, panel 6) were normalized to protein concentrations as determined by the BCA assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135725#sec002" target="_blank">Materials and Methods</a>).</p

Rapid, Precise, and Accurate Counts of <i>Symbiodinium</i> Cells Using the Guava Flow Cytometer, and a Comparison to Other Methods

<div><p>In studies of both the establishment and breakdown of cnidarian-dinoflagellate symbiosis, it is often necessary to determine the number of <i>Symbiodinium</i> cells relative to the quantity of host tissue. Ideally, the methods used should be rapid, precise, and accurate. In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer. We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument's automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate. We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.</p></div