ena-STUDY-UNIVERSITY OF ZURICH-25-05-2021-14:37:14:285-414
Fluorescence in situ hybridization for the detection of planktonic freshwater ciliates
Progress in molecular analytical tools, such as environmental high throughput sequencing (HTS) techniques, opened new insight into ciliate assemblage structures and dynamics in aquatic ecosystems. However, for exact determination of abundances, the classical morphology-based method QPS (quantitative Protargol staining) is still the most reliable way. Nonetheless, the whole procedure of QPS, as well as the subsequent evaluation are very time consuming and analyzed sample volumes are rather small. CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) appears to be an optimal method for analyses of planktonic ciliates. Larger sample volumes at high sampling frequencies can be evaluated with species-specific probes for even closely related, similar looking as well as very tiny ciliates. We designed 13 species- and genera-specific oligonucleotide probes for several planktonic ciliates. The CARD-FISH protocol was modified and successfully applied on single cell cultures and mock assemblages. A comparison to QPS-counts proved the quantitative character of this new method for the determination of species abundances. First tests on field samples suggest that CARD-FISH could become a promising new methodological approach for the determination and quantification of planktonic ciliates.
Progress in molecular analytical tools, such as environmental high throughput sequencing (HTS) techniques, opened new insight into ciliate assemblage structures and dynamics in aquatic ecosystems. However, for exact determination of abundances, the classical morphology-based method QPS (quantitative Protargol staining) is still the most reliable way. Nonetheless, the whole procedure of QPS, as well as the subsequent evaluation are very time consuming and analyzed sample volumes are rather small. CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) appears to be an optimal method for analyses of planktonic ciliates. Larger sample volumes at high sampling frequencies can be evaluated with species-specific probes for even closely related, similar looking as well as very tiny ciliates. We designed 13 species- and genera-specific oligonucleotide probes for several planktonic ciliates. The CARD-FISH protocol was modified and successfully applied on single cell cultures and mock assemblages. A comparison to QPS-counts proved the quantitative character of this new method for the determination of species abundances. First tests on field samples suggest that CARD-FISH could become a promising new methodological approach for the determination and quantification of planktonic ciliates.