1. health and fitness
2. disease

Coral Microbiota
What kind of microorganisms?
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Corals have been found to harbor a wide variety of microbes:
Heterotrophic eukaryotes
Bacteria
Archaea
Viruses
The majority of studies thus far have centered on bacteria
associated with coral.
• Coral microbiota 16S rRNA sequences were dominated mostly
by bacteria (80–90%), with both healthy and bleached corals
harboring similar dominant taxa.
What kind of advantages?
• There is increasing evidence that coral microbiota are
crucial to at least two aspects of the host’s physiology:
• Biogeochemical cycling (cycling carbon, fixing nitrogen,
chelating iron).
• Pathogen resistance (producing antibiotics).
• Toxicity (only in few bacteria)
What kind of detection method?
• Sampling of bacterial rRNA genes amplified from nucleic
acid provide high taxonomic resolution for environmental
samples (Olsen et al. 1986).
• While cultivation-based approaches provide important
information on the metabolism of some microorganisms.
• The vast majority (>99%) of marine microorganisms do
not grow on enriched media (Azam 1998).
How is the diversity of bacteria?
• Altogether, more than 250 hemolytic bacterial isolates were
identified which belong to:
• Firmicutes (Bacillus cereus group)
• Gammaproteobacteria (Acinetobacter & Vibrio)
• Actinobacteria (the genera Brevibacterium)
Another study
• A bacterial origin of PTX has been suggested by Frolova et
al. (2000) based on results from a PTX sensitive
immunoassay.
• Following screening of 420 isolates obtained from toxic
samples of Palythoa colonies:
• The gram-negative Aeromonas and Vibrio species were
identified to produce compounds antigenically related to
PTX.
Are the bacteria specific for coral?
• These clades are widespread in aquatic environments and are often
associated with other organisms either as symbionts or as pathogens.
• Many marine animals in close association with zoanthid colonies:
• Sponges
• Soft corals
• Gorgonians
• Mussels
• Crustaceans
• Predators of the zoanthids: polychaete worms, starfish and fish
• MAY contain high concentrations of PTX and exhibit remarkable toxin
tolerance (Mebs 1998; Gleibs and Mebs 1999).
Some example?
• Vibrio harveyi and V. tubiashi are pathogens of fish and
invertebrates.
• V. gallicus lives in a symbiotic relationship with the abalone
Haliotis tuberculata (Sawabe et al. 2004).
• Other representatives of the genus Vibrio are known to
synthesize the neurotoxin tetrodotoxin (Yu et al. 2004;
Wang et al. 2008).
• This toxin shares some ecological similarities with PTX in
that it is also widely distributed in diverse marine
organisms and is produced by several distinct clades of
bacteria (Wang et al. 2008).
What is the PTX?
• PTX is considered to be one of the most potent marine
compounds because of its extremely high toxicity in
mammals with a LD50 of 10–100 ng kg-1.
• The toxin was primarily detected in marine zoanthids of
the genera Palythoa, Protopalythoa and Zoanthus (Moore
and Scheuer 1971; Gleibs et al. 1995; Gleibs and Mebs
1999) as well as in the dinoflagellate Ostreopsis siamensis
(Ukena et al. 2001).
How does the PTX act?
Palytoxin acts through the Na-K-ATPase of cell
membranes by inducing pore formation which results in
breakdown of the transmembrane potential (Habermann
1989; Artigas and Gadsby 2003; Hilgemann 2003; Louzao
et al. 2008).
Since the Na-K-ATPase is a crucial membrane component
of animal cells, its particular interaction with PTX explains
the highly lethal potency of the toxin.
Whereas chemical synthesis of palytoxin was successfully
accomplished (Suh and Kishi 1994), its biosynthetic origin
is still not entirely clear.
Site and sample collection
• Date
• Geographical Site
• Depth (7 to 10 m)
Measuring the water quality parameters:
• The concentration of phosphate, nitrate, ammonia and
nitrite.
• The biological demand of oxygen and the total amount of
solids in suspension.
• Conductivity
• pH
• Salinity
• Temperature
Mucus sampling and DNA extraction
• The mucus will drain of the coral samples using a sterile
syringe on board of the boat.
• These samples were immediately placed in an ice box
and brought to the laboratory.
• Sea water of the surrounding environment nearby the
coral will collect in a sterile bottle (A total of 1 liter).
DNA extraction
• The mucus DNA was extracted using the Fast DNA Spin
for Soil Kit (QBiogene, Vista, CA, USA).
16S rDNA amplification, cloning & sequencing
• Three samples (water, healthy coral and diseased coral mucus)
Universal primers:
• 27F (5-AGA GTT TGA TCM TGG CTC AG-3 )
• 1492R (5-TAC GGY TAC CTT GTT ACG ACT T-3 ).
Thermocycler conditions:
• 95 C for 3 min (1 cycle)
• 95 C for 1 min
• 51 C for 1.5 min
• 72 C for 3 min (25 cycles)
• 72 C for 30 min (1 cycle)
• PCR were performed 5 times for each sample.
• Electrophoresis through 1% agarose gel.
• Fragments with 1.4kb were gel excised and purified using
• UltraClean DNA Purification Kit (MoBio, Carlsbad, CA, USA)
Thank you
for your attention!