Little more than a decade ago, toxic phytoplankton monitoring with DNA probes was still in the realm of science fiction. Today it is a reality. Probes for some key micro-algae are already in commercial use and ultimately DNA probes could become a major HAB monitoring tool.

What exactly are they?

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DNA probes have been in use for more than a decade in medicine and dentistry, but they have only recently been developed with the aquaculture industry in mind. The DNA of a selected micro-alga is sequenced, and the sequence of nucleotides that makes up its genetic code is compared with that of other related algae using readily available computer software packages. Unique nucleotide "signatures" (usually between 15 and 50 nucleotides) are selected to develop into probes. The ribosomal RNA (rRNA) of the cell is targeted because it is abundant in cells and so good recognition, or a good "hit", is ensured. The probes will have some "tag", for example fluorescein, attached for easy recognition of a positive assay. The probes can be prepared for a number of different assay systems, but require intensive trialling to ensure there is no cross-reactivity with other micro-algae species.

Pseudo-nitzschia and Alexandrium whole cell format DNA probes are now used routinely in New Zealand's phytoplankton monitoring programmes as an aid to risk management decision-making, and the monitoring laboratory (Cawthron Institute) is approved by International Accreditation New Zealand (recognised under ISO17025).

How are they used?

Identification of micro-algae to species level is vital so that shellfish growers, and health and industry officials, can make safe and economically sound harvesting decisions.

Standard phytoplankton monitoring methods involve identifying toxic species in a water sample and counting them. It is labour intensive and requires expert training in the recognition of the critical algae. In some case scanning electron microscope preparation of samples can be required to differentiate species. This can be expensive and cause delays. DNA probes offer an exciting and rapid alternative.

Whole cell hybridisation

The probes can be prepared for use against whole cells using a filter system and in this case the probes will have a dye attached, eg. FITC or Texas Red. Following cell fixation, addition of probes and hybridisation at pre-determined temperature and salinity, the cells can be viewed on the filter under an epifluorescence microscope using a fluorescein band-pass filter set.

This group are difficult to differentiate to species level under the light microscope, and include highly toxic and non-toxic species. Shellfish farmers and public health officials are keen to know which species are present to guide them in developing a risk assessment. The assay takes 4 hours, and full phytoplankton counts and identifications with a risk assessment are with the clients within 24 hours of receipt of sample.

Sandwich hybridisation assay (SHA)

One drawback of the "whole cell" DNA probes is the need for expensive microscopes and technical expertise. Some micro-algae are also delicate and don't "fix" well for these assays. A recent DNA probe development, the sandwich hybridisation assay, is faster, partially automated, and utilises an enzyme-driven colour reaction, which can be quantified using a micro-plate reader. The rapid assay (1 hour turn-around) uses pre-prepared probes and assay plates which minimise labour and reduce the need for scientific expertise. The suite of probes can be prepared to suit client needs and the assay can be performed in the laboratory as well as aboard ships. The resultant colour reaction can be correlated to cell numbers and quantified on a plate reader.

Cawthron Institute now offers probes for the diatom genus Pseudo-nitzschia commercially.

The next step …

Chris Scholin, MBARI, is currently working on real-time data collecting stations, based on the SHA, which will be moored on site. Real time data sets of temperature, salinity, toxic algae, etc. will be relayed back to on shore stations for analysis and report generation. Prototype systems are already well down the track.

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