Using the ‘Snot sucker’ method to discover bacterial assemblages within compartments of the coral holobiont

It is accepted that corals have a diverse, and host-species specific microbiota; however it is poorly documented as to how they are organised within the coral holobiont. A number of studies have been carried out, all using a variety of techniques such as crushed coral, scraping of surface, airbrushing, swabs and milked mucus to name a few, which, although they have given some fascinating results are not highly regarded by the authors of this article as they believe that these methods are easily contaminated. For this reason, when trying to understand the differences, and organisation of assemblages within the coral holobiont, they used a new method for removing layers of bacterial habitats within the coral naming the apparatus used the ‘snot sucker’.

Corals were collected, mounted on a screw cap system and returned to the reef until collection. Sample collection took place at a reef flat by Heron Island (23^o27’S, 151^o55’E).Samples of the surrounding water column and sediment were also collected as a potential supply of coral associated bacteria.

The snot sucker is a ‘50ml falcon tube, with two 3-way valves grafted onto it, one at the bottom and one at the top. A 60ml syringe, with tubing, was attached to the bottom stopper valve allowing filtered water to be flushed over the coral and loosely attached surface mucus layer (SML) collected by the top valve.’ The snot sucker was used both in situ and in the lab, and previously used methods, mentioned earlier (airbrushed-tissue layer, collection of tissue slurry etc.) were tested by the authors too.

DNA fragments were visualised using DGGE with Sybr ® Gold and a UV transilluminator. Results were statistically analysed using a one-way permutation analysis of variance (PERMANOVA), analysis of contribution to similarities (SIMPER), and results are laid over a 2-D plot using a non-metric multidimensional scaling (MDS) and based on a similarity profile (SIMPROF). They looked for differences in both, bacterial assemblages within compartments of the coral holobiont and also the techniques used. From the Shannon-Weiner diversity index, the diversity varied significantly between different techniques; the snot sucker being the greatest in diversity and swabs and milked mucus being the lowest. The worst technique employed appeared to be the swabs, with average similarity between that and other sample types seldom exceeding 25%; they came to the conclusion that it may include contamination from other sources as there was a 35% similarity to sediment for example.

The study demonstrated that bacterial communities do differ between compartments of the holobiont; with significant differences being shown between all the coral compartments and the surrounding environment samples; supporting the idea that coral harbour and maintain a distinct microbial flora. Within their discussion the authors explain where possible sources of bacteria, which may colonise the SML arrive from e.g./ faecal matter, water column etc. and how the fact that the SML is significantly different in bacterial diversity compared to the surrounding environment showed that bacterial communities will occur even on what may be considered as the harshest environment offered by the coral, as it is constantly affected by hydrodynamic processes and the chemical make-up differs from coral to coral.

A review of:

Sweet, M. J., Croquer, A. And Bythell, J. C. (2010). Bacterial assemblages differ between compartments within the coral holobiont. Coral Reefs. Vol. 30. (1) pp. 39-52.

Found at: http://www.springerlink.com/content/e26284260u337u4h/export-citation/

Accessed on: 22/12/2011

Marvellous Mucus

Many coral reefs are found in nutrient poor zones, but are none-the-less high primary production ecosystems. Mucus secreted by some corals (mucopolysaccharide) carry energy and nutrients to a range of planktonic and benthic consumers, and supports a large microbial growth within the reef system.The conductors of this study decided to investigate which bacteria in seawater are favoured by the release of freshly detached mucus material. Short-term changes in organic carbon and nitrogen, and microbial community composition were simultaneously analysed in mucus enriched seawater.

Coral mucus and seawater were collected from the lagoon of Dahab, in the southern part of the Gulf of Aqaba, northern Red Sea, in May 2004. Polyps of one of the most common Red Sea scleractinian corals (Fungia sp.) with a diameter of 5-10cm were collected from depths of 3-8m, and then exposed to air for 3 minutes in order to trigger mucus production. Mucus produced in the first minute was discarded to reduce contamination of bacteria from the coral surface and mucus produced in the further 2 minutes was aseptically collected and bottled. The coral mucus was homogenised and mixed with seawater (1:10) obtained from the same site within 60 minutes and bottled; bottles of seawater which hadn’t been enriched with mucus were kept for control. All bottles were kept incubated for 50 hours at in situ temperature (24^oC) and light conditions (500-800 µmol quanta m^-2day^-1) at a depth of 1m.

Each bottle was portioned and filtered onto precombusted GF/F filters. The concentrations of particulate organic C (POC) and particulate N (PN) and stable isotope ratios of C to N were determined using dried filters with THERMO NA 2500 elemental analyser coupled to THERMO/Finnigan MAT Delta plus isotope ratio mass spectrometer. The POC and PN concentrations were two- to threefold higher in the mucus amended incubations when compared with the controls.

15ml samples were taken after 0, 2, 4, 6, 10, 26 and 50 hours for bacterial cell counting and fluorescent in situ hybridisation (FISH). Portions were filtered onto polycarbonate membrane filters, after 24h of fixation in buffered paraformaldyhide solution. Total cell numbers were determined by staining with 4’, 6-diamino-2-phenylindole and automated epifluorescence microscopy. The initial cell numbers in the mucus-seawater mixture were marginally higher than the control cell numbers. Additions of mucus lead to an almost fourfold increase in bacterial abundance after 10 hours; whereas the control approximately doubled over the whole 50 hours.

Addition of mucus to the seawater clearly favoured the growth of the Gammaproteobacteria. The relative abundance of these microorganisms initially developed similarly in the mucus and control incubations. Between 4 and 10h, the proportions of Gammaproteobacteria in the mucus incubations rose steeply, to around 60%after 10h and around 70% toward the end of the experiment. The Gammaproteobacteria never exceeded 40% of the total cell counts in the controls.

A review of:

Allers, E., Niesner, C., Wild, C., and Pernthaler, J. (2008). Microbes Enriched in Seawater after Addition of Coral Mucus. Applied and Environmental Biology. Vol. 74 (10). p. 3274-3278