Visualising symbiotic bacteria: a practical application of fluorescence in situ hybridisation (FISH)

Many marine microbiologists use fluorescence in situ hybridisation (FISH) to identify and visualise specific bacteria. Cells are permeabilized to allow probes to enter, once inside the cell the probes target specific complementary nucleic acid sequences and become hybridized to them. The probes are labelled with fluorescence which can then be analysed using epifluroscence, laser scanning microscopy or flow cytometry. Typically when researchers use this technique to study symbiotic bacteria the tissue in question is paraffin or frozen sectioned; the former technique involves lengthy preparation by trained and skilled individuals whilst the later often compromises resolution. Authors of the current paper developed a FISH procedure which allows entire gill filaments to be used. The preparation of which simply requires careful dissection techniques and the ability to follow an off the shelf FISH kit, further more using this technique a single gill filament would correspond to the same area as 80 paraffin or frozen sections.

The authors of this paper were specifically interested in the symbiosis between chemosynthetic bacteria and two species of deep-sea mussels. The technique they developed using a whole mounted gill filament allowed them to visualise the the spatial distribution of thiotrophic and methanotrophic bacteria for which they had developed specific probes for. They found that in both species the majority of the lateral surface contained bacteriocytes for both thiotrophic and methanotrophic bacteria; however cilary cells did not contain bacteriocytes. In one species this was visualised by dark band-like zones which did not fluoresce which were the ciliary junctions, in another dark non-fluorescing oval areas were seen which corresponded to ciliary tufts on the gill. Authors predict that using this technique in addition to confocal laser-scanning a three dimensional analysis could also take place.

I feel the authors of this paper have taken the logical step forward in science in order to use the FISH technique to its full potential which has allowed them to gather more information about the specific microbial community they were interested in. Visualisation of the spatial distribution of microbial communities provides information on both community structure as well as possible insights into ecological function. It therefore makes perfect sense to examine larger areas at any one time, not to mention the amount of time it saves compared to looking at tiny 10µm thick sections. I agree with the authors in their prediction that this technique could be used for other species with symbiotic bacterial communities and possibly even for multi-layered tissues if the correct permeability conditions were optimized.

Fujinoki, M., Koito, T., Fujiwara, Y., Kawato, M., Tada, Y., Hamasaki, K., Jimbo, M., et al. (2012). Whole-mount fluorescence in situ hybridization to visualize symbiotic bacteria in the gills of deep‑sea mussels. Aquatic Biology, 14(2), 135–140.

http://www.int-res.com/abstracts/ab/v14/n2/p135-140/

An example of a well written post from 2011

Small Cells, Big Trouble. Toxic Mercury Bioaccumulation

Mercury is used by marine bacteria in demethylation metabolic reactions and results in the formation of two forms of methylmercury (MeHg), both toxic to humans. MeHg concentrations have been rising in coastal waters as increasing amounts of mercury are deposited into the sea either by polluted rivers or atmospheric contamination from coal-fired power stations. Organisms consume microbes contaminated with MeHg, which is easily absorbed but difficult to excrete due to it being more soluble in lipids than water. The result is an increasing build-up of MeHg as it moves through the food web, from bacteria to fish (biomagnification). If contaminated fish (e.g. top predators like tuna) are consumed by humans it can have a number of detrimental effects to health. The developing nervous systems of unborn babies are at particular risk.MeHg levels in coastal regions have been well reported. However, a number of recent studies have shown that oceanic levels are also increasing, even though such areas are far away from potential mercury contamination sources.

Heimburger et al (2010) conducted a 20 month study at a site in the North-western Mediterranean, chosen because it is separated from coastal waters by the Ligurian current whilst being relatively close to the shore (~ 50 km). They measured the MeHg concentrations and the distribution of phytoplankton throughout the water column, down to the sea floor (2350 m). MeHg concentrations showed a double peak of 0.22 pM and 0.82 pM at depths of approximately 50 m (euphotic zone) and 400m (aphotic zone) respectively. The upper euphotic zone (0-10m) showed the lowest concentrations (< 0.10 pM). The levels changed seasonally, with the highest peaks in the autumn months correlated with blooms of nano- and picophytoplankton in the euphotic zone during oligotrophic conditions.

The authors concluded that the high MeHg concentrations at 50 m in the autumn were caused by the higher surface to volume ratio of the small nano- and picophytoplankton cells compared to larger photosynthetic microbes found nearer the surface. Thet speculated that this enabled a higher rate of mercury intake and mercury methylation. The even higher concentration in the aphotic zone was attributed to the slow sinking of small POM aggregates from the nano- and picophytoplankton reaching the deeper water where heterotrophic microbes feed upon them, starting the MeHg accumulation. ,Despite having less input of mercury (mainly atmospheric) than coastal areas, the oligotrophic conditions of the open ocean seem to favour mercury methylation as nano- and picophytoplankton are more common.

I reviewed this article because it possibly has a link with my earlier blog about a shift in marine phytoplankton to smaller sized species as a result of global warming. This coupled with high amounts of atmospheric release of mercury from power stations, could increase the amount of MeHg biomagnification in marine organisms caught for human consumption.

Source: Heimburger L, Cossa D, Marty J, Migon C, Averty B, Dufour A and Ras J (2010) Methyl mercury distribution in relation to the presence of nano- and picophytoplankton in an oceanic water column (Ligurian Sea, North-western Mediterranean). Geochimica et Cosmochimica Acta. 74: 5549-5559.

(Edited from a post by Lee Hutt, 2011)

2 comments:

Samantha Bowgen said...

It wouldn't be as realistic in the open water, but maybe if mercury pollution was treated in coastal waters, this would reflect well on the amount dispersed out to sea.
Do you know if there are any current methods in place to treat mercury contaminated water? I have read about successful small scale treatments. E.g. where doses of Stannous chloride were used, successfully removing >94% of mercury. Although the solution may then cause problems of its own..

Chemicals can be used to render the contaminating mercury insoluble. Or even are there treatments to reduce the activity of the microbes involved..

Most likely not sensible ideas, but would be interesting to see how mercury pollution is currently being tackled.

4 April 2012 13:48

Lee Hutt said...

Hi Sami

As far as I am aware there are not any activley used methods in treating this problem, in the ocean anyway. There seems to be several studies looking into this but most seem to focus on fresh water or soil.

Zhuang et al (2003) used lignin (derivative from trees) to remove mercury from soil and water. Because it contains many polar functional groups it can remove metal ions by binding with them and creating large macromolecule lignin-metal complexes, eliminating the metal availability. I think the problem with this however is what other metals does it eliminate from the environment? Could cause more harm than good. Other techniques I have read about involve electrodes but I doubt this would be realistic in an ocean setting.

Zhuang JM, Walsh T and Lam T (2003) A new technology for the treatment of mercury contaminated water and soils. Environmental Technology. 24: 897-902.

7 April 2012 09:46

Welcome to the BIOL3309 Blog Site

Most of your learning about recent developments will come from reading and evaluating the latest research papers. The coursework mark will be derived from tutor assessment of your online postings to the blog site, which should show evidence of current awareness of the scientific rationale and potential applications of recent research findings relevant to the module content. The assessment will be based on the frequency and — most importantly — the quality of your contributions (both your own reviews and the comments on other people’s contributions). 

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