Thomas A. Richards

Across eukaryotes, the nuclear envelope exhibits distinct remodelling strategies during mitosis: complete breakdown (open mitosis), partial disruption (intermediate), or full retention (closed). However, both light and electron microscopy of the nuclear envelope, at sufficient resolution to determine integrity and mode of remodelling, have proven technically challenging. Here, Concanavalin A (ConA), a glycan-binding plant lectin, provides discernible nuclear envelope/endoplasmic reticulum (NE/ER) labelling in Ultrastructure Expansion Microscopy (U-ExM). Applied post-expansion, ConA circumvents antibody optimisation and labels the NE across mammalian cells and diverse microbial eukaryotes. By imaging four opisthokont and amoebozoan species with established mitotic strategies, C. perkinsii, D. discoideum, S. pombe, and S. arctica, we distinguished the full spectrum of NE remodelling. Applied to species with poorly characterised mitotic strategies, ConA revealed intermediate mitosis with polar fenestrae in the multinucleate stramenopile A. limacinum and a life cycle-coupled switch between open and closed mitosis in the amoebozoan P. polycephalum. These observations support the hypothesis that multinucleated life cycles favour closed or intermediate mitosis, protecting chromosomes from capture by neighbouring spindles during synchronous divisions. Together, these results establish ConA in combination with expansion microscopy as a broadly applicable tool for uncovering the diversity and evolution of NE remodelling across eukaryotes.

Endosymbiosis is a major driver of evolutionary innovation and underpins the function of diverse ecosystems. The origins and evolution of endosymbiosis are challenging to study experimentally due to the short-lived culturability of many microbial strains derived from endosymbiotic interactions. The facultative endosymbiosis between the ciliate, Paramecium bursaria, and the green alga, Micractinium conductrix (Chlorellaceae, Trebouxiophyceae), is ecologically widespread and has emerged as a powerful lab-tractable model system. This endosymbiosis is founded upon a reciprocal nutrient exchange, but each of the species can be cultured independently enabling quantification of symbiotic fitness effects, new partnerships to be generated in the lab, and co-associations to be subject to experimental evolution. To date, evolve-and-resequence approaches have been limited due to a lack of high-quality genome assemblies enabling gene variants to be identified. Here, we report a near telomere-to-telomere genome assembly for M. conductrix 186b, using a range of sequencing technologies. Comparative analysis shows that this is one of the most complete Chlorellaceae algal genome assemblies available to date. To aid accurate gene calling and annotation, we conducted both RNAseq and Iso-Seq transcriptome sequencing experiments. Collectively, these ‘omics datasets will facilitate: (i) comparative genomics studies of endosymbiont evolution, (ii) evolve-and-resequence experiments, (iii) genome-scale metabolic modeling studies, and (iv) identification of targets for genetic modification experiments and biotechnological applications.

Microbial host–parasite interactions shape global biogeochemical cycles, yet their cellular and evolutionary mechanisms remain poorly understood. Here we developed a model pathosystem to explore the interaction between the eukaryotic microparasite, Pirsonia diadema , and its host, the bloom-forming diatom, Coscinodiscus . Culture conditions recapitulated Pirsonia ’s rapid life cycle in vitro, and live-cell imaging enabled the quantitative analysis of infection dynamics and host mortality. Genome sequencing and time-resolved dual RNA sequencing of Pirsonia revealed unique genetic innovations relative to their oomycete relatives and an upregulation of an expanded multi-gene family of integrin-like proteins in Pirsonia zoospores during infection. Targeted drug perturbations combined with ultrastructure expansion microscopy confirm stage specific roles for Pirsonia ’s cytoskeleton, including actin-dependent host attachment and formation of intracellular parasite-induced feeding structures and microtubule-associated processes in zoospore differentiation. Together, these findings reveal mechanisms underpinning Pirsonia infection and provide insights into the evolution of strategies that underpin parasitic shunts in marine trophic networks.

Multiple displacement amplification (MDA) enables whole-genome amplification from single cells, but introduces chimeric artifacts that severely compromise downstream analyses, particularly with long-read sequencing. Here, we systematically evaluate long-read PacBio HiFi sequencing of MDA amplified DNA from single cells using the model green alga Chlamydomonas reinhardtii. We show that MDA-derived libraries exhibit highly uneven coverage and extreme chimera rates impacting up to 70% of reads, leading to thousands of artefactual structural variants and misassemblies when assembled using algorithms designed for bulk sequencing. To overcome these challenges, we developed lrSAGA (long-read Single Amplified Genome Assembly), a novel tool to assemble long-read MDA sequencing datasets. Assemblies generated using lrSAGA are more complete, more contiguous, and have 75-95% fewer misassemblies compared to conventional assembly algorithms. Although overall contiguity is limited by MDA coverage dropouts, we demonstrate that up to 68% of the C. reinhardtii genome can be accurately assembled from just a single haploid cell. We further validated lrSAGA using published Oxford Nanopore and PacBio HiFi data from single or half Caenorhabditis elegans worms, generating accurate and highly complete assemblies. Applying our approach to single protist cells isolated from environmental water samples, we performed PacBio HiFi single-cell genome sequencing of four uncultivated microbial eukaryotes: an amoeboflagellate from the Naegleria genus, a flagellate from the Bodo genus, and two deep-branching flagellates from the enigmatic CRuMs supergroup, Collodictyon triciliatum and Diphylleia rotans. From single cells, we generated high-quality draft genome assemblies estimated to be 70-84% complete, demonstrating the potential of long-read single-cell genomics to unlock genome diversity from uncultivated microbial eukaryotes.

Parasites of the Perkinsea lineage infect both unicellular eukaryotic and metazoan hosts. The Pathogenic Perkinsea Clade (PPC) has been associated with Severe Perkinsea Infections (SPI) and mortality events in wild tadpole populations across the US and in captive European Tree Frogs (Hyla arborea) in the UK. To investigate the spread of this emergent disease and identify additional putative parasites, we launched a UK-based citizen science project called ‘Tadpole Doctor’. Volunteer participants collected tadpoles, sediment samples from ponds suspected to contain tadpoles, or alternatively, sediment from ponds where there was evidence of recent loss of a tadpole population. In addition to the UK samples, tadpoles with SPI symptoms were collected in Alaska and Florida, along with asymptomatic tadpoles in the Czech Republic. To identify traces of PPC and related lineages, we developed a semi-targeted long-read SSU-ITS rDNA sequencing approach, revealing an unprecedented molecular diversity of the wider Perkinsea clade NAG01, where PPC branches in SSU rDNA phylogenies. Seven groups of sequences other than PPC were found associated to tadpole livers, potentially reflecting novel parasitic infections. We also identified three sister groups to the PPC clade, among which one group was found in a tadpole, and another was frequently encountered in environmental DNA (eDNA) metabarcoding surveys. Deeper characterisation of this diversity may identify new threats to amphibians. Overall, these data illustrate how citizen-based research can be useful for assessing organismal distribution patterns, enabling further targeted studies, while also raising public awareness of the vulnerability of amphibians to infectious disease.

Recurrent transitions in feeding strategies have shaped the eukaryotic tree of life, as unrelated groups independently evolved similar solutions to common ecological challenges. One of the most interesting yet poorly studied of these shifts is the transition towards osmotrophy. We reconstructed the evolution of four eukaryotic groups that specialized in osmotrophy through convergent evolution. Here we show that these groups arose most likely during the Tonian period (1,000–720 million years ago) or slightly before, and possess a genetic toolkit enriched in shared metabolic functions. We report signatures that are compatible with horizontal gene transfer encompassing at least 20% of this toolkit. Phylogenetic reconciliation analyses show that this fraction of the toolkit ranks in the upper percentiles for inferred horizontal gene transfers, particularly in the period in which the osmotrophic groups originated. Moreover, analyses of the total gene content using supervised phylogenetic screening identified 166 gene tree topologies that are supportive of transfer events involving distantly related eukaryotic osmotrophs. These data include transfer highways between Fungi and Pseudofungi and between Labyrinthulea and Teretosporea. Our work thus unravels the evolutionary history of four independent transitions towards specialization in osmotrophy within the eukaryotes, supporting a role of gene transfer in the evolution of these groups.

Bodo is a cosmopolitan genus of free-living bacterivorous single-celled flagellates in the class Kinetoplastea. Genus Bodo is considered the closest free-living lineage to the parasitic lineages Trypanosoma and Leishmania , the causative agents of the human diseases sleeping sickness, Chagas disease and leishmaniasis. Currently, a single genome exists for the one formally described species in the genus, Bodo saltans . Previous studies on B. saltans have shown that it is dependent on an endosymbiotic bacterium from the order Holosporales, ‘ Candidatus Bodocaedibacter vickermanii’. Using single-cell sequencing, we isolated, sequenced and assembled genomes for seven uncultured Bodo spp. cells from a freshwater sample from Royal Leamington Spa, UK. Using comparative genomics, we show that these seven cells represent three potentially novel Bodo species exhibiting unexpected levels of diversity at the genome level. Our results indicate that small subunit ribosomal DNA sequencing, often used to classify Bodo flagellates, is insufficient for determining species delimitation in this genus. In addition, we recovered a Holosporales bacterium genome from all seven Bodo spp. cells. Surprisingly, these seven endosymbionts also represent three novel species, congruent with the phylogeny of the host and exhibiting lineage-specific adaptations. This diversity and host–symbiont association would be indistinguishable in routinely used metabarcoding or bulk sequencing pipelines, thus demonstrating the power of single-cell sequencing to reveal diversity within lineages of microbial eukaryotes.

Endosymbiosis played a key role in the evolution of cellular complexity, but mechanisms underpinning establishment and maintenance remain unclear. Paramecium bursaria harbours Chlorellaceae algal endosymbionts and is a valuable model system for understanding photosymbiosis. However, there are a lack of molecular tools available for exploring the association. Here we report stable transformation of the P. bursaria endosymbiont Micractinium conductrix SAG 241.80. Bioluminescent reporter assays were used to identify endogenous regulatory sequences for transgene expression and to refine a protocol for delivery of exogenous DNA. The bleomycin resistance gene Shble was then identified as an effective selectable marker for isolating transformed cell lines. We demonstrate that endogenous introns enhance transgene expression and that isolation of transformants with desirable characteristics can be achieved without extensive screening by using a 2A peptide to couple expression of an upstream non-selectable transgene to expression of the Shble selectable marker. Finally, we demonstrate that M. conductrix SAG 241 . 80 transformants expressing fluorescent proteins can be introduced into host cells and observed in the absence of selection, enabling distinct endosymbiont genotypes to be unambiguously identified, compared and monitored within the host environment. The development of molecular tools reported here opens new avenues for addressing unresolved questions in photosymbiosis. Significance Statement: Paramecium bursaria is a single-celled, mixotrophic ciliate which forms photosymbiotic interactions with Chlorellaceae green algae and is a popular model system for symbiosis research. We established a stable transformation protocol for the P. bursaria algal endosymbiont M. conductrix SAG 241.80 and identified properties of synthetic gene constructs which facilitate isolation of transformants with robust transgene expression that can be detected within the host environment. This marks significant progress in model system development and opens new avenues for exploring molecular and cellular mechanisms underpinning photosymbiotic interactions.

Summary: Akiyoshi, 2025 presented a hypothesis with implications for the early evolution of eukaryotes and eukaryotic cell division machinery. In this Correspondence, the authors conclude that this hypothesis leads to a highly implausible scenario.

Endosymbiosis is a major driver of evolutionary innovation and underpins the function of diverse ecosystems. The origins and evolution of endosymbiosis are challenging to study experimentally due to the short-lived culturability of many microbial strains derived from endosymbiotic interactions. The facultative endosymbiosis between the ciliate, Paramecium bursaria, and the green alga, Micractinium conductrix (Chlorellaceae, Trebouxiophyceae), is ecologically widespread and has emerged as a powerful lab-tractable model system. This endosymbiosis is founded upon a reciprocal nutrient exchange, but each of the species can be cultured independently enabling quantification of symbiotic fitness effects, new partnerships to be generated in the lab, and co-associations to be subject to experimental evolution. To date, evolve-and-resequence approaches have been limited due to a lack of high-quality genome assemblies enabling gene variants to be identified. Here, we report a near telomere-to-telomere genome assembly for M. conductrix 186b, using a range of sequencing technologies. Comparative analysis shows that this is one of the most complete Chlorellaceae algal genome assemblies available to date. To aid accurate gene calling and annotation we conducted both RNAseq and Iso-Seq transcriptome sequencing experiments. Collectively these omics datasets will facilitate: i) comparative genomics studies of endosymbiont evolution, ii) evolve-and-resequence experiments, iii) genome-scale metabolic modelling studies, and iv) identification of targets for genetic modification experiments and biotechnological applications.