A CARD-FISH protocol for the identification and enumeration of cyanobacterial akinetes in lake sediments

Akinetes are the dormant cells of Nostocales (cyanobacteria) that enable the organisms to survive harsh environmental conditions while resting in bottom sediments. The germination of akinetes assists the dispersal and persistence of the species. The assessment of the akinete pool in lake sediments is essential to predict the bloom formation of the Nostocales population. We present here the implementation of an improved catalysed reporter deposition (CARD)-fluorescence in situ hybridization (FISH) protocol to assist the identification and quantification of akinetes in sediment samples. Several 16S rRNA gene oligonucleotide probes were evaluated for labelling akinetes of various species of Anabaena, Aphanizomenon and Cylindrospermopsis. Akinetes of all the taxa studied were successfully labelled and could be easily detected by their bright fluorescence signal. The probes' specificity was tested with 32 strains of different taxa. All six Cylindrospermopsis raciborskii strains were labelled with a specific probe for its 16S rRNA gene. A more general probe labelled 73% of the Anabaena and Aphanizomenon strains. The counting data of field samples obtained with CARD-FISH and the regular light microscopy approach did not differ significantly, confirming the suitability of both methods. The CARD-FISH approach was found to be less time-consuming because of better visibility of akinetes.