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Single-cell transcriptomics has the potential to provide novel insights into poorly studied microbial eukaryotes. Although several such technologies are available and benchmarked on mammalian cells, few have been tested on protists. Here, we applied a microarray single-cell sequencing (MASC-seq) technology, that generates microscope images of cells in parallel with capturing their transcriptomes, on three species representing important plankton groups with different cell structures; the ciliate Tetrahymena thermophila, the diatom Phaeodactylum tricornutum, and the dinoflagellate Heterocapsa sp. Both the cell fixation and permeabilization steps were adjusted. For the ciliate and dinoflagellate, the number of transcripts of microarray spots with single cells were significantly higher than for background spots, and the overall expression patterns were correlated with that of bulk RNA, while for the much smaller diatom cells, it was not possible to separate single-cell transcripts from background. The MASC-seq method holds promise for investigating "microbial dark matter", although further optimizations are necessary to increase the signal-to-noise ratio.

Studying microbes from a single-cell perspective has become a major theme and interest within the field of aquatic microbiology. One emerging trend is the unfailing observation of heterogeneity in activity levels within microbial populations. Wherever researchers have looked, intra-population variability in biochemical composition, growth rates, and responses to varying environmental conditions has been evident and probably reflect coexisting genetically distinct strains of the same species. Such observations of heterogeneity require a shift away from bulk analytical approaches and development of new methods or adaptation of existing techniques, many of which were first pioneered in other, unrelated fields, e.g., material, physical, and biomedical sciences. Many co-opted approaches were initially optimized using model organisms. In a field with so few cultivable models, method development has been challenging but has also contributed tremendous insights, breakthroughs, and stimulated curiosity. In this perspective, we present a subset of methods that have been effectively applied to study aquatic microbes at the single-cell level. Opportunities and challenges for innovation are also discussed. We suggest future directions for aquatic microbiological research that will benefit from open access to sophisticated instruments and highly interdisciplinary collaborations.

High-throughput sequencing-based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go-to method for studying microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in applying long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics data sets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.

High-throughput sequencing-based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go-to method for studying microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in applying long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics data sets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.

Phagotrophic protists are key players in aquatic food webs. Although sequencing-based studies have revealed their enormous diversity, ecological information on in situ abundance, feeding modes, grazing preferences, and growth rates of specific lineages can be reliably obtained only using microscopy-based molecular methods, such as Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). CARD-FISH is commonly applied to study prokaryotes, but less so to microbial eukaryotes. Application of this technique revealed that Paraphysomonas or Spumella-like chrysophytes, considered to be among the most prominent members of protistan communities in pelagic environments, are omnipresent but actually less abundant than expected, in contrast to little known groups such as heterotrophic cryptophyte lineages (e.g., CRY1), cercozoans, katablepharids, or the MAST lineages. Combination of CARD-FISH with tracer techniques and application of double CARD-FISH allow visualization of food vacuole contents of specific flagellate groups, thus considerably challenging our current, simplistic view that they are predominantly bacterivores. Experimental manipulations with natural communities revealed that larger flagellates are actually omnivores ingesting both prokaryotes and other protists. These new findings justify our proposition of an updated model of microbial food webs in pelagic environments, reflecting more authentically the complex trophic interactions and specific roles of flagellated protists, with inclusion of at least two additional trophic levels in the nanoplankton size fraction. Moreover, we provide a detailed CARD-FISH protocol for protists, exemplified on mixo- and heterotrophic nanoplanktonic flagellates, together with tips on probe design, a troubleshooting guide addressing most frequent obstacles, and an exhaustive list of published probes targeting protists., QC 20210415

Heterotrophic nanoflagellates (HNF) are considered as major planktonic bacterivores, however, larger HNF taxa can also be important predators of eukaryotes. To examine this trophic cascading, natural protistan communities from a freshwater reservoir were released from grazing pressure by zooplankton via filtration through 10- and 5-mu m filters, yielding microbial food webs of different complexity. Protistan growth was stimulated by amendments of five Limnohabitans strains, thus yielding five prey-specific treatments distinctly modulating protistan communities in 10- versus 5-mu m fractions. HNF dynamics was tracked by applying five eukaryotic fluorescence in situ hybridization probes covering 55-90% of total flagellates. During the first experimental part, mainly small bacterivorous Cryptophyceae prevailed, with significantly higher abundances in 5-mu m treatments. Larger predatory flagellates affiliating with Katablepharidacea and one Cercozoan lineage (increasing to up to 28% of total HNF) proliferated towards the experimental endpoint, having obviously small phagocytized HNF in their food vacuoles. These predatory flagellates reached higher abundances in 10-mu m treatments, where small ciliate predators and flagellate hunters also (Urotricha spp., Balanion planctonicum) dominated the ciliate assemblage. Overall, our study reports pronounced cascading effects from bacteria to bacterivorous HNF, predatory HNF and ciliates in highly treatment-specific fashions, defined by both prey-food characteristics and feeding modes of predominating protists.