Megan Gross

Quantifying abundances of unicellular eukaryotes (protists) remains a central challenge in microbial ecology, as methodological differences can strongly influence abundance estimates and ecological interpretation. Although molecular tools have thus far greatly improved our understanding of protists, high rRNA gene copy numbers limit quantitative inferences. Digital PCR (dPCR) has emerged as a promising tool for absolute quantification, yet its application for unicellular eukaryotes and its comparability to established cell‐based methods remain insufficiently explored. Here, we develop species‐specific dPCR assays for two important freshwater ciliates ( Urotricha castalia and Urotricha pseudofurcata ) and establish gene copy number correction factors to enable highly accurate quantitative abundance estimates. We assess assay performance using controlled laboratory experiments and apply the approach to environmental samples, directly benchmarking dPCR against catalyzed reporter deposition‐FISH (CARD‐FISH). Under controlled conditions, dPCR and CARD‐FISH yielded comparable accuracy, with dPCR showing superior precision. In field applications, method‐dependent differences emerged, reflecting both methodological constraints and biological variability. Notably, dPCR provided an overall higher sensitivity, enabling robust detection of low‐abundance taxa. Our results highlight dPCR as a scalable and sensitive approach that, when combined with appropriate correction strategies, represents a significant step towards more reliable molecular quantification of protists. At the same time, differences between methods underscore the value of integrating molecular and microscopy‐based approaches. We propose that combining dPCR with tools such as CARD‐FISH can offer complementary insights into protist population dynamics. Such integrative frameworks provide a powerful path forward for improving abundance estimates and advancing quantitative microbial ecology.

Microorganisms play key roles in ecosystem functioning, making reliable methods for assessing their dynamics essential. Advances in molecular technologies now enable their quantification in environmental samples based on DNA marker genes. Among these, digital PCR has emerged as a powerful tool for detecting and quantifying organisms based on their gene copies, with numerous platforms that are currently available. However, these platforms differ in their underlying technologies, and comparative studies that evaluate the performance and reproducibility remain limited. Here we compared different platform parameters across the QX200 digital droplet PCR from Bio-Rad and the QIAcuity One nanoplate-based digital PCR from QIAGEN. We used synthetic oligonucleotides and DNA extracted from varying cell numbers of the ciliate Paramecium tetraurelia and additionally tested the impact of two restriction enzymes on gene copy number quantification. Both platforms demonstrated similar detection and quantification limits and yielded high precision across most analyses. We found a general tendency of higher precision using the HaeIII restriction enzyme instead of EcoRI, especially for the QX200 system. Gene copy number estimates from ciliate DNA were reproducible between platforms and showed a linear trend for an increasing number of cells for both platforms. These findings highlight the importance of cross-platform evaluations to ensure robust and reproducible gene copy number analysis in unicellular eukaryotes and support a potential broader application of digital PCR in environmental monitoring studies.

Short-branch Microsporidia were previously shown to form a basal grade within the expanded Microsporidia clade and to branch near the classical, long-branch Microsporidia. Although they share simpler versions of some morphological characteristics, they do not show accelerated evolutionary rates, making them ideal candidates to study the evolutionary trajectories that has led to long-branch microsporidians unique characteristics. However, most sequences assigned to the short-branch Microsporidia are undescribed, novel environmental lineages for which the identification requires knowledge of where they can be found. To direct future isolation, we used the EukBank database of the global UniEuk initiative that contains the majority of the publicly available environmental V4 SSU-rRNA gene sequences of protists. The curated OTU table and corresponding metadata were used to evaluate the occurrence of short-branch Microsporidia across freshwater, hypersaline, marine benthic, marine pelagic, and terrestrial environments. Presence-absence analyses infer that short-branch Microsporidia are most abundant in freshwater and terrestrial environments, and alpha- and beta-diversity measures indicate that focusing our sampling effort on these two environments would cover a large part of their overall diversity. These results can be used to coordinate future isolation and sampling campaigns to better understand the mysterious evolution of microsporidians unique characteristics.