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An Analysis of the Microbial Community Associated with the Mucus of Ringed Coral (Montipora patula)

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Finally, calm. A short swim from shore and a new world opens around me. Above the ocean surface, there is chaos. Cars rush, factories pollute, plastic is littered, waves pound violently. Just below the surface, the world is calm. Sounds are muffled; only the rhythmic clicks of microscopic shrimp and the in-and-out breaths through my snorkel distract from the seemingly infinite stretch of water on all sides. Fish nip at algae on the coral, unaware of my presence. Along the shore, the marine and the terrestrial worlds collide. The intersection here presents a multifaceted relationship that is increasingly crucial to the health and longevity of both worlds.

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Hawaii coastline near the sample site.


Although peaceful to the senses, the ocean mirrors the constant turmoil above. Coral reefs lay at the very core of this chaos. Because they provide habitat for high levels of biodiversity, they are crucial to maintaining the delicate balance between producers and consumers in the coastal marine environment.

I was born and raised in Hawaii; I grew up around the ocean, camping and snorkeling and exploring the tidal pools. I have been snorkeling since I could swim. I recently visited my favorite childhood beach and was taken aback by the murky quality of the water and the lack of wildlife in the area. There were far more people camping than there had been in the past, and I began to realize that the worldwide degradation of coral reef ecosystems had hit home, literally. The decline in coral population, overall reef health, and marine biodiversity has recently accelerated, with links to human-induced causes such as overexploitation, overfishing, increased sedimentation, and nutrient overloading. Natural disturbances include temperature extremes, flooding, and predatory outbreaks. In response to the sharp increase of these stressors, coral bleaching, which occurs when the level of zooxanthellae in the tissues of polyps increases or declines sharply, has become more frequent and widely distributed worldwide (Buchheim 1998).

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This diagram shows the location of coral mucus as a barrier between the coral animal and the water column, where potential diseases usually originate.


After witnessing the degradation of Hawaii’s marine ecosystems during my childhood, I began to do research and talk to local marine biologists about different aspects of the progression of coral health decline. Studies concerning the unique and delicately balanced relationship between zooxanthellae and the coral animal have shown that it may be the most ecologically important relationship on the reefs. Zooxanthellae reside within the coral tissue and are photosynthetic, providing nutrients to the polyp in the form of fixed carbon, with the coral providing carbon dioxide and shelter in return (Buchheim 1998). Bacterial communities within the mucopolysaccharide layer of the coral animal may play equally crucial roles, but studies concerning these communities are much less extensive (Knowlton and Rohwer 2003). Coral mucus is an ecotome, providing the boundary between the coral animal and the surrounding water column, the source of potential invasive microbes (Ritchie 2006). All corals secrete mucus, which coats the outside of the entire organism. I formulated a project to identify the microbial community of coral mucus because I learned that it is a critical but understudied link in the recent onset of widespread coral disease. This study was intended to provide baseline data for future studies involving coral disease and its devastating impact on marine ecosystems worldwide.

Some of the prokaryotic organisms in the mucopolysaccharide layer may protect corals from pathogens either by blocking their entry into the coral, or by producing antibiotics, or by enhancing the ability of corals to defend themselves against predators or competitors (Knowlton and Rohwer 2003). Describing the species composition of the microbial community within the mucopolysaccharide layer of coral is the first step in studying the function of coral mucus as a barrier against diseases that may cause harm to corals.

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The Montipora patula coral.


In the same way that human mucus protects the body from harmful bacteria and viruses, coral mucus has implications to serve as a similarly protective layer. Before large-scale impact studies can be conducted on the implications of mucus’s role in coral disease, the microbial community associated with coral mucus must be studied. The microbial community of the common Hawaiian coral species Montipora patula was analyzed through the application of three distinct methods: gram staining, fluorescence in situ hybridization (FISH), and ribotype analysis. Three different identification assays were chosen to provide a more complete description of the microbial communities within M. patula mucus. The 16S rRNA sequences allowed for the taxonomic identification of the microbial community. FISH was utilized to classify and give information on relative abundance of the community based on taxa-specific molecular probes. Gram staining was used to indicate the presence and abundance of both gram-positive and gram-negative bacteria, while also providing an insight into the mucus structure.

Methods

Mucus Extraction

Extracting mucus from the coral is referred to as “milking” the coral. Two samples of M. patula three centimeters in diameter were collected from Leleiwi Beach in Hilo, Hawai’i, at a depth of roughly eight meters. Upon collection, samples were immediately placed on ice in individual bags for transport. Samples were rinsed with 0.2 l filtered seawater (FSW), and then placed tissue side-down in glass funnels in separate beakers. Two 15-mL falcon tubes were filled with the resulting mucus-water solution and centrifuged at 4,200 rpm for 14 minutes. The supernatant was removed, resulting in concentrated mucus slurry, which was used for all subsequent procedures.

Gram Staining

Common gram-staining protocol (adapted from Benson’s Microbiological Applications) was applied to slides with 100 µL of mucus aliquoted out onto slides with three wax-drawn paraffin wells. The slides were dehydrated in a drying oven set at 70˚C, and then were stained with crystal violet for 20 seconds, Gram’s iodine for 60 seconds, decolorized with ethyl alcohol and finally safarin for 60 seconds, with a rinse of deionized water between each application. The slides were blotted dry with bibulous paper, labeled, and left to air-dry. All slides were stored at 4˚C and analyzed with a light microscope.

DNA Extraction, Amplification, Cloning, and Sequencing
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This was taken during the ribotype analysis to ensure that the PCR procedures had worked to amplify a fragment of the desired length (around 1,500 bp). The first number in the lane label is the sample number and the second is the elution number.


A Qiagen DNeasy animal tissue kit was used according to the manufacturer’s protocol for all DNA extractions. DNA extraction protocol was followed for each of the two coral samples individually, using 250 µL of mucus slurry from each. The polymerase chain reaction (PCR) protocol was acquired from Rasche et. al. using the primers 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1520R (5’-AAGGAGGTGATCCAGCCGCA-3’). For PCR cleanup, MoBio PCR Ultra Clean PCR purification kit was used according to the manufacturer’s protocol, and cloning was completed using the Promega T-gem easy vector cloning kit. The cloned plasmids were then prepped for sequencing according to the Promega Wizard SV Plasmid Purification System protocol. From the cloned colonies, 24 were picked for liquid growout and subsequent plasmid purification. Samples were grown out in 5 mL of liquid LB culture media and incubated at 37˚C for 24 hours. Samples were sequenced at the University of Hawaii at Manoa using an Applied Biosystems 377XL DNA sequencer.

Fluorescence in situ Hybridization (FISH)

Fluorescence in situ hybridization began with mucus fixed in 4% paraformaldahyde 3X PBS, and then pelleted by centrifugation at 5,000 g for 15 minutes. The pellet was then resuspended in 1% PBS (0.5 mL). After 500 µL of ice-cold absolute ethanol was added, the mucus was stored at -20˚C until slide preparations. Into each of eight hand-drawn paraffin wells, 30 µL of pelleted mucus was aliquoted, and dried overnight in a sterile hood. The hybridization solution was formulated using 360 µL of 5 M NaCl, 40 µL of 1 M Tris/Hcl, 2 µL of 10% SDS, 700 µL of formamide and 898 µL of autoclaved milli-Q water, made to a final volume of 2 mL per slide. Slides were then dehydrated with three one-minute rinses in ethanol. Of the four wells per slide, one well on every slide was a control, and the remaining three wells were hybridized with a single probe per well, at a 1:8 ratio of probe to hybridization solution. The four probes used were beta proteobacteria (BAT), gamma proteobacteria (GAM), all eubacteria (EUB), and low GC content (gram negative, LGC). Control sections were hybridized without any probes. Slides were then hybridized in a hybridization oven for two hours at 46˚C. Following hybridization, slides were washed with warm (48˚C) buffer (35% formamide, 0.08M NaCl, 0.5M EDTA, 1% SDS) and rinsed with cold (4˚C) distilled deionized water, dried, and stored at -20˚C until analysis when cover slips were mounted on the hybridized slides with fluorescent mounting media and then viewed and photographed using an epiflourescent microscope.

Data Analysis And Discussion

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Gram-stained mucus (at 100x magnification) indicated presence of both gram-positive (stained purple) and gram-negative (stained red) bacteria.


Each set of data collected from the FISH, gram staining, and cloning procedures were assessed for identification of the bacteria. Gram-stained slides were viewed and photographed using a light microscope, and the structure of the mucus was analyzed. Relative abundance of gram-positive and gram-negative bacteria was noted. Sequences resulting from ribotype analysis procedures were viewed and trimmed to eliminate the vector using the Sequencher computer program. These sequences were then input into the Basic Local Alignment Search Tool online (BLAST, www.ncbi.nlm.nih.gov) for bacterial matches. The first (usually uncultured) match and the first match of known taxonomy were both recorded for every sample, making note of the ecological role of each. The same web site was then used to discern functions or associations, based on previous published studies in the database. Data from the ribotype analysis were graphed according to bacterial division.

The gram-staining results rendered useful insight into the structure of coral mucus. Both gram-positive and gram-negative indicators were found in the stained mucus, but approximately 95% of the stained mucus appeared red (indicating gram-negative bacterial presence) and about 5% of the stained mucus was purple, indicating gram-positive bacterial presence. A significant benefit of the gram staining was that it allowed for the complexities of the mucus structure and composition to be viewed and analyzed. Although some bacterial cells were identified, the more interesting results of the gram-staining procedures showed that the mucus is structurally complex, with small, dense, plus-shaped regions surrounded by streaks of linear formations of the mucus, which follow the same trend in a vertical direction.

Of the 37 samples that were sequenced, 35 came back with readable results. These 35 results are represented in the Bacterial Abundance graph.

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Figure 3: Frequency of bacterial types within the mucus were found through the 16s ribotype analysis procedures. This graph shows that the majority of the microbial community within M. patula’s mucus is alpha-proteobacteria.


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Figure 4: Bacterial species identified through ribotype analysis were researched and those found to have known associations with marine organisms or with the marine environment were documented as shown. These data serve as implications toward the function of different microbes within the coral mucus.


 

Of these species identifications, those with significant links to known marine organisms or the marine environment are displayed in the Species Analysis Table. The results of the fluorescent in situ hybridization were not conclusive because very few bacterial cells were actually identified under the fluorescent microscope. The methodology of this segment of the experiment may have been flawed as a result of degraded fluorescent probes. 

The combination of the three separate identification assays was utilized with the intention to provide a fairly thorough assessment of the microbial community associated with M. patula. Of the 35 16S sequences obtained, 16 were found to be closely related to known associates of other marine invertebrates. Some of the microbes were determined to be known symbionts of marine sponges and deep-water marine tubeworms, as well as other corals. Two identified microbes were known to be associates of the microbial community of Muricea elongata, a coral species found in the Caribbean, while two others were known associates of Oculina patagonica, a common coral species in the Mediterranean.

Gram staining provided valuable insight into the structure of the mucopolysaccharide layer, information that could prove to be crucial in studying what functions mucus serves for the coral animal and how it is specifically designed to do so. Gram-negative bacterial presence (stained red on slides) was overwhelming in comparison to the roughly 5% of purple-stained gram-positive bacteria.

Conclusion

Further research would include studying the function of each type of bacteria identified so that their role in the mucus and in relation to the coral animal can be assessed. It has not been concluded whether mucus-associated microbial communities are species-specific, but Rohwer et. al. determined in 2002 that microbial communities associated with the entire coral animal are known to be species-specific. Thus, with implications toward coral conservation throughout Hawaii and the ocean abroad, extended research on separate species of coral should be conducted.

I hope that my work on this project will one day provide critical baseline data toward effective coral conservation and prevention of coral disease. Coral mucus is an understudied but integral element of this crisis, but with findings such as those I have provided with this study, our understanding of the way coral animals are dealing with the onslaught of disease and other stressors will strengthen and provide a means for more effective coral conservation.

Living in Hawaii and spending so much time around the ocean has contributed so much to my life: It has made me who I am. I aspire to reverse the negative relationship between the marine and the terrestrial environments, and this project is an embodiment of that aspiration.

Bibliography

Baron, Samuel. ”Surface Layers.” Medical Microbiology. University of Texas. Retrieved from the World Wide Web on 9 February 2008. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mmed.section.289

Brown, Alfred E. Benson’s Microbiological Applications, 10th ed . Boston: McGraw Hill, 2007, 95-96.

Buchheim, Jason. “Coral Reef Bleaching.” Odyssey Expeditions. 1998. Retrieved from the World Wide Web on 7 January 2008. http://www.marinebiology.org/coralbleaching.htm

Coral Microbial Ecology. U.S. Geological Survey, 2005. Retrieved from the World Wide Web. http://coastal.er.usgs.gov/coral-microbes/

Guppy, Reia, and John C. Bythell. “Environmental effects of bacterial diversity in the surface mucus layer of the reef coral Montastraea faveolata.” Marine Ecology Progress Series 328 (2006).

Knowlton, Nancy, and Forest Rohwer. “Multispecies microbial mutualisms on coral reefs: The host as a habitat.” American Naturalist 162 (2003): S51-S62.

Lesser, Michael P., Charles H. Mazel, Maxim Y. Gorbunov, and Paul G. Falkowski. “Discovery of symbiotic nitrogen-fixing cyanobacteria in corals.” Science 305.5686 (13 August 2004): 997-1,000.

Rasche, Frank, et al. “Rhizosphere bacteria affected by transgenia potatoes with antibacterial activities compared with the effects of soil, wild-type potatoes, vegetation stage and pathogen exposure.” FEMS Microbiology Ecology 56 (2006): 219-235.

Ritchie, Kim B., and Garriet W. Smith. “Microbial communities of coral surface mucopolysaccaride layers.” In: E. Rosenburg and Y. Loya, eds. Coral Health and Disease. New York: Springer, 2004.

Ritchie, Kim B. “Regulation of microbial populations by coral surface mucus and mucus-associated bacteria.” Marine Ecology Progress Series 322 (2006): 1-14.

Rohwer, Forest, V. Seguritan, F. Azam, and N. Knowlton. “Diversity and distribution of coral-associated bacteria.” Marine Ecology Progress Series 243 (2002): 1-10.

Wegley, Linda, et al. “Metagenomic analysis of the microbial community associated with the coral Poreites astreoides.” Environmental Microbiology 9.11 (2007): 2,707-2,719.

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