Uncultivated microbial eukaryotic diversity: a method to link ssu rRNA gene sequences with morphology

Area of Science:

• Microbiology
• Molecular Biology
• Eukaryotic Cell Biology

Background:

• Traditional protist classification relies on cultivation and morphology, but cultivation-independent methods yield novel sequences (phylotypes) lacking morphological context.
• Linking newly discovered rRNA phylotypes to known morphological descriptions is crucial for accurate taxonomic classification.

Purpose of the Study:

• To develop and demonstrate methods for combining rRNA-targeted fluorescent in situ hybridization (FISH) with immunostaining to link genetic sequences with cellular morphology in uncultivated protists.
• To establish a high-throughput approach for characterizing novel protist lineages.

Main Methods:

• Whole-cell rRNA-targeted FISH using eukaryote or ciliate-specific probes.
• Combined FISH with cytoskeletal immunostaining (anti-α-tubulin, phalloidin for actin) or organellar staining (Mitotracker®, anti-Hsp70 for hydrogenosomes).
• Applied methods to environmental samples to link rRNA phylotypes to specific morphological features.

Main Results:

• Successfully linked rRNA phylotypes to microtubule structures, describing flagellate and ciliate morphology in diverse environments.
• Connected *Naegleria* spp. to their amoeboid form using actin staining.
• Linked uncultivated ciliates to morphologically similar *Colpoda*-like ciliates via tubulin immunostaining.

Conclusions:

• Combining rRNA-targeted FISH with immunostaining provides a rapid and efficient method for linking genetic data with morphological features of uncultivated protists.
• This integrated approach complements and validates sequence data, facilitating the description of novel protist lineages and improving taxonomic understanding.