A new fluorescent oligonucleotide probe for in-situ identification of Microcystis aeruginosa in freshwater

Cyanobacteria colonize different environments and blooms can occur in both contaminated and non-contaminated water bodies (freshwater, brackish and marine areas). Among 150 known cyanobacteria genera, more than 40 species are able to produce toxins. The latter are natural compounds, which differ from both a chemical and toxicological point of view and are responsible for both acute and chronic poisoning in animals and humans. Among the main classes of cyanotoxins, microcystins are frequently found in the environment. Fast and accurate methods for unequivocally identifying microcystin-producing cyanobacteria, such as Microcystis aeruginosa in water bodies, are necessary to distinguish them from other non-toxic cyanobacteria and to manage and monitor algal blooms. For this purpose, we designed, developed and validated an oligonucleotide probe for FISH (Fluorescence In Situ Hybridization) analysis to detect Microcystis aeruginosa even at relatively low concentrations in freshwater with the intention of adding it to the µAQUA microarray for freshwater pathogens, which had only genus level probes for Microcystis on the array field tested in this EU project. The FISH probe was designed using the ARB software with the Silva database in the framework of the MicroCoKit project. We tested various fixative methods to minimise the natural autofluorescence from chlorophyll-a and certain accessory pigments (viz., phycobilins and carotenoids). The FISH probe (MicAerD03) was tested on pure cultures of Microcystis aeruginosa, and then applied successfully to water samples collected from different sampling points of the River Tiber (Italy), using a laser confocal microscope. Subsequently, the probe was also conjugated at the 5’ end with horse-radish peroxidase (HRP-MicAerD03) to apply the CARD-FISH for increasing the fluorescence signal of the mono-fluorescently labelled probe and make it possible to detect M. aeruginosa using an epifluorescence microscope. Samples taken within the project MicroCokit indicated that the microarray signals for Microcystis were coming from single cells and not colonial cells and we confirmed this with the CARD-FISH protocol used here that was used to validate the microarray signals detected at the genus level in that project. This paper provides a new early warning tool for investigating M. aeruginosa even at low cell concentrations in surface water, which can be added to the microarray from µAqua for all freshwater pathogens to complete the probe hierarchy for Microcystis aeruginosa.JRC.D.2-Water and Marine Resource