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Oligonucleotide probes are increasingly being used to characterize natural microbial assemblages by Tyramide Signal Amplification-Fluorescent in situ Hybridization (TSA-FISH, or CAtalysed Reporter Deposition CARD-FISH). In view of the fast-growing rRNA databases, we re-evaluated the in silico specificity of eleven bacterial and eukaryotic probes and competitor frequently used for the quantification of marine picoplankton. We performed tests on cell cultures to decrease the risk for non-specific hybridization, before they are used on environmental samples. The probes were confronted to recent databases and hybridization conditions were tested against target strains matching perfectly with the probes, and against the closest non-target strains presenting one to four mismatches. We increased the hybridization stringency from 55 to 65% formamide for the Eub338+EubII+EubIII probe mix to be specific to the Eubacteria domain. In addition, we found that recent changes in the Gammaproteobacteria classification decreased the specificity of Gam42a probe, and that the Roseo536R and Ros537 probes were not specific to, and missed part of the Roseobacter clade. Changes in stringency conditions were important for bacterial probes; these induced, respectively, a significant increase, in Eubacteria and Roseobacter and no significant changes in Gammaproteobacteria concentrations from the investigated natural environment. We confirmed the eukaryotic probes original conditions, and propose the Euk1209+NChlo01+Chlo02 probe mix to target the largest picoeukaryotic diversity. Experiences acquired through these investigations leads us to propose the use of seven steps protocol for complete FISH probe specificity check-up to improve data quality in environmental studies.

Microcystin (MC) is a common and widespread toxin which represents a health hazard to humans and animals. MC toxin concentrations are monitored by various direct or proxy techniques (HPLC, LC-MS/MS, ELISA, PPIA), however, these techniques do not discriminate producing species from non-producing ones. In order to simultaneously provide the identity and activity of cyanotoxin producing species in freshwater lakes, we applied simple, and fully detailed, whole cell fluorescent in situ hybridization enhanced by tyramid signal amplification (TSA-FISH). DNA oligonucleotide probes MICR3 and MCYA were targeting 16S rRNA and mcyA-mRNA, respectively. The mcyA gene is coding for the MC synthetase enzyme involved in MC synthesis. Controls were acquired with the general eubacterial 16S rRNA probe EUB338, for TSA-FISH assay, and standard HPLC and LC-MS/MS as standard methods for the measurements of MC concentration. Results obtained from monoclonal strains and natural samples demonstrated a specific identification of Microcystis species and were able to discriminate MC producing from non-producing ones. In addition, the MCYA probe allowed the specific detection of MC-synthetase mRNA within Planktothrix isothrix (Oscillatoriale) filaments. Two kinds of mcyA-mRNA labeling were observed in these cells, spots like and plasmid like, which illustrates the well-known plasticity of microbial genome to adapt to environmental stresses. We demonstrated that a simple TSA-FISH assay allows acquiring rapidly dual information of the presence and abundance of potentially toxic species, while identifying species actively producing MC-synthetase mRNA, a proxy of MC toxin. This technique has the potential to be developed into an effective environmental monitoring tool. In addition, detail visualization of cellular mRNAs is powerful for the acquisition of ecological and biomolecular studies of toxic cyanobacteria.