Previously, researchers have been using epifluorescence microscopy protocols established for seawater when trying to determine how abundant bacteria are within mucus layers of corals. These procedures, however, are inappropriate due to the viscosity and autofluorescent properties of coral mucus layers. This paper explores a variety of different methods to solve this problem and allows us to study ecological interactions within these microenvironments.
Samples of coral and surrounding seawater was taken from seven different sites along two transects on the east side of Maui, Hawaii. Each transect, one north and one south, began at a freshwater seep. Samples were taken from the seep itself, the nearest living coral to the seep and corals that were around 50m west from each seep. The mucus layer of Porites lobata at depths between 1 to 5 meters and the seawater directly 0.5m above the coral were sampled. Only seawater was taken from the seep sites as there was no living coral present.
Samples taken from three Porites lobata colonies were used for the dispersal experiment. Three different treatment methods were used with 1ml of 1:10 dilutions of coral mucus. The most successful method by far was to disassociate the cells from the mucus by incubating it within 0.4% of trypsin enzyme for 15 minutes to degrade bonds between the cells and basement membranes and bonds between cells. This was followed by 5ul ml-1 DAPI staining, filtration and mounting onto slides with a vectasheild mixed with NAO to counter stain lipids. It was also found that it was best not to quench the autofluorescence of the mucus as when this was attempted the zooxanthellae could not be seen.
Bacterial abundance and cell size were also determined from the samples of both the coral mucus and the surrounding water. As expected, the abundance of bacteria within the coral mucus layer was much higher than that in the surrounding water. However, the bacteria within the surrounding sea water were much larger than those within the coral mucus. The frequency of dividing cells was much higher within the sea water also. This provokes interest as the zooxanthellae, which are more than 4 orders of magnitude larger than bacteria, enrich the mucus environment. This shows evidence that the bacteria are unable to utilise this carbon source for growth. Alternatively there may be size-selective grazing pressures present or that zooxanthellae are less ‘leaky’ than other dinoflagellates. It could also suggest that that the excess nutrients provided by the seeps are stimulating growth in a higher proportion than first expected.
The authors are unable to answer many questions that arise from these results as it is among the first reports of zooxanthellae concentration in coral mucus. However, it does set out a protocol which is a good starting point to develop a tool box of consistent methods that will allow microbial ecology hypotheses to be challenged in a viscous, autofluorescent mucus environment.
A review of : Garren,M; Azzam,F; (2010); New Method for Counting Bacteria Associated with Coral Mucus; App.Env.Micro.; 76(18); 6128-33.
1 comment:
Theodora - an interesting paper. Because of the emphasis on development of a simple method for obtaining direct counts of bacteria in coral mucus, one might overlook some very surprising results. Firstly, the fact that so many zooxanthellae were present in the mucus. I think they say that the biomass of zooxanthellae was much greater than that of bacteria. Do they discuss this at all? Presumably, theses corals are not bleaching, so why would they be shedding zooxanthellae? The fact that mucus bacteria were much smaller than seawater bacteria seems very surprising also. In the paper reviewed by Josh, it was shown that mucus stimulated growth of gamma proteobacteria. These are presumably copiotrophs, which from other studies we would expect to much bigger than oligotrophs growing in seawater with lower nutrients. Could antibiotic action by some members of the community be responsible? I wonder how they explained these unexpected results?
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