iDiv Annual Conference 2025

Correcting the Molecular Bias: Optical High Throughput and Gene Copy Number Calibration for Realistic Protistan Abundance Estimates

30 Sept 2025, 16:30

15m

Lecture Hall XXII (Audimax)

Talk Molecular Biodiversity and Evolution Molecular Biodiversity and Evolution

Speaker

Dr Nadine Möbius (FSU Jena, Institute of Biodiversity, Ecology and Evolution, German Center for Integrative Biodiversity Research (iDiv) Halle Jena Leipzig)

Description

Protists are key players in microbial food webs, yet their role in groundwater ecosystems remains understudied—partly due to limitations in quantifying their abundances using molecular methods. A major challenge lies in the highly variable number of 18S rRNA gene copies across taxa, which can lead to a drastic overestimation of the quantitative role of specific taxa in protistan communities.
We present an integrated approach that combines Multispectral Imaging Flow Cytometry, qPCR, and 18S rRNA gene amplicon sequencing to improve the quantification and characterization of protistan communities. Protistan monocultures ranging from 3 to 60 µm in size were used to build a reference image library for convolutional neural network-based classification, achieving over 85% recognition precision. This enables not only automated counting but also the potential observation of feeding events. However, image-based classification is limited to trained taxa and cannot capture the full diversity of natural groundwater communities.
To identify potential key players beyond the reference set, molecular data remain essential. To account for the high variability in gene copy numbers, however, we defined taxon-specific correction factors. For instance, Ciliophora isolates harbored on average 5.4 × 10³ (Glaucoma sp.) or 7.9 × 10⁵ (Colpidium sp.) 18S rRNA gene copies per cell, while the cercozoan Cercomonas sp. contained only 1.5 × 10¹ copies per cell. This emphasizes the need for taxon-specific correction factors in molecular community analyses to avoid overestimations spanning several orders of magnitude.
Our approach provides a framework for more realistic protistan abundance estimates in groundwater where small flagellates such as Cercozoa may dominate trophic interactions. In turn, the ecological role of ciliates may be far less prominent than suggested by uncorrected molecular data. This altogether allows for a more accurate assessment of the groundwater food web structure.