Rapid and ultra-sensitive fluorescent test for the tracking of toxic algae in the marine environment (EU Project DETAL)
With the DETAL project it was intended to develop a rapid, sensitive, and easy to use detection method for harmful marine microalgae. The detection is based on sample filtration and subsequent whole cell hybridization. The cells are fluorescently labeled by antibody and / or oligonucleotide staining and detected and enumerated using a solid phase cytometer.
The main advantage of this method is the specific detection of whole cells, which allow not only cell counts, but even the evaluation of the results by microscopy. The final goal is the adaptation and use of this method for routine monitoring of toxic algae with a high sample throughput.
In the molecular genetics groups at the AWI we designed rRNA targeting oligonucleotide probes for different taxonomic groups of algae. Beside a number of probes developed in other projects (see MICROPAD), in DETAL we focus on genera and species of the Prymnesiophyceae (haptophyta), Pseudo-nitzschia (diatoms), and Alexandrium (dinoflagellates). For the automated detection, the FISH protocol will be adapted for the use in combination with a laser scanning solid phase cytometer (Chemscan RDI, Chemunex). For a more detailed description of the cytometer see http://www.chemunex.com/products/chemframe.htm.
Introduction to harmful algal blooms (HAB) and their impacts
Although algal "blooms" can be a common occurrence in aquatic habitats, they can also impart serious detrimental effects to the environment and to public health when they are associated with toxicity for human and/or sea products. Before national fisheries can proceed with any intensified exploitation of the coastal marine waters for food production, the extent of harmful algal blooms (HABs), in time and space, must be reduced or monitored more effectively.
The occurrence of HABs has been known since antiquity, but during recent years these events have become an increasing problem in coastal marine waters, and particularly along European coasts. This phenomena is increasing not only in terms of the frequency of their occurrence but also in the severity of their economic impact. Monitoring for HABs is a requirement of EU directive 91/492/CEE and most European countries have monitoring programmes to ban shellfish harvesting during harmful algal bloom events or when shellfish reach a specified level of toxicity that is potentially harmful to humans. The microalgal species causing harmful algal blooms (HABs) are relatively few in number (200) in comparison to the total biodiversity of microalgal species believed to exist (>10,0000), and they are distributed among all major taxonomic algal groups: diatoms, dinoflagellates, haptophytes, and cyanobacteria (blue-green algae) and are found on a global scale.
Planktonic toxic blooms can cause neurotoxic shellfish poisoning (NSP), paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), ciguatera fish poisoning (CFP) or diarrhetic shellfish poisoning (DSP). New species of harmful algae are continuously detected, and more worrisome new toxins being found and chemically characterised. Currently, PSP is the most widespread shellfish poisoning occurring all over the world, followed by DSP. These two toxic syndromes are of particular importance on European coasts. The other poisonings, ASP and NSP, have more restricted geographical occurrences, although there are now new reports of ASP from Scotland. CFP is only localised in tropical waters.
Monitoring with high sample throughput and low detection limit by applying molecular methods, the aim of the DETAL project
Molecular biological tools have greatly enhanced our ability to identify toxic algae species/strains rapidly and with higher sensitivity and to estimate distribution of species in time and space. DNA and RNA probes, cell-surface antibodies and other molecular and biochemical techniques can be used collectively to verify and cross-corroborate identifications made using classical procedures. Although rRNA and antibody probes are available for some toxic algae, a platform that allows for a routine automated application of these probes to identify HAB species in field and laboratory studies is not yet available (Tyrrell et al. 1997). As many monitoring programs are based on cell counts, the use of whole cell hybridisations for the specific detection and the combination with an automated counting system seems to be promising.
Our objectives were:
- to adapt the protocols for antibody and oligonucleotide staining to the automated detection in a solid phase cytometer and thereby
- to develop a new automated method for the rapid and sensitive detection of selected harmful algal species in the marine environment;
- to validate the specificity of the methodology on cultured and field material;
- to transfer this technology to the different national harmful algal monitoring networks which represent the best controlled sampling conditions existing to study the evolution of the populations of toxic algae.
Fluorescence in situ hybridisation in combination with solid phase cytometry
We use CARD (catalysed reporter deposition)-FISH , or synonymously called TSA (tyramide signal amplification)-FISH, for the hybridisation of microalgae in combination with solid phase cytometry (SPC). In this assay the catalytic activity of the enzyme horseradish peroxidase (HRP) is applied. The oligonucleotide probe is labelled with HRP, which binds to its substrate, a fluorescing tyramide (fluorescein labelled in SPC), when it is immobilised through binding of the probe to its target. This results in a signal amplification up to 20 times. The signal amplification step enables a better discrimination between non-target autofluorescing microalage or particles and so only positive labelled microalgae with a high fluorescence intensity are counted by the SPC.
After FISH the filter is analysed by SPC. In SPC, the laser is moved over cells immobilised onto a solid support and allows the rapid enumeration of several thousand cells within three minutes. The scanned filter is then analysed by microscopy, whereby each positive data point is automatically moved to each positve datapoint by the computer software. Solid phase cytometry is especially useful for the detection of rare events with a detection limit of one cell per filter. We adapted SPC in combination with FISH in our group for the detection of (toxic) microalgae.
Differentiation of algal species with similar morphologies in a fluorescence in situ hybridisation
Two algal species with similar cell morphology can be distinguished by fluorescence in situ hybridisation, using two probes labeled with different fluorochromes. A mixture of Emiliania huxleyi and Prymnesium nemamethecum cells was hybridised with a mixture of a universal eukaryotic probe (EUK1209R), Cy5 labeled, and a probe specific for the genus Prymnesium (PrymGl01A), FLUOS labeled. The image shows the overlay of three detection channels (Confocal Laser Scanning Microscope), where the colours are defined as follows: green = FLUOS, blue = Cy5, red = autofluorescence. The Prymnesium nemamethecum cells are stained with both probes and have therefore a signal in green and blue (=> turquoise cells). The Emiliania huxleyi cells are only stained with the eukaryotic probe and are therefore blue.