High-quality reference genome assemblies have become essential for deepening our understanding of biodiversity, yet obtaining them for many species remains surprisingly challenging. Drawing on experiences from the European Reference Genome Atlas (ERGA) community, we focus on permit and sample-handling procedures leading up to nucleic acid sequencing, covering tasks such as ensuring ethical and legal compliance, verifying accurate species identification, maintaining sample integrity during transport, and isolating high-quality DNA or nuclei. While many of the challenges and solutions we discuss are broadly relevant, our regulatory and logistical examples are primarily from Europe. By synthesising practical guidance, we highlight the crucial importance of taxonomic expertise, proper vouchering and biobanking, rigorous cold-chain management or alternative preservation methods, and emphasise adherence to packaging and shipping requirements for biological materials. We showcase examples spanning diverse regions, taxa and source materials, which underscore the importance of context-specific strategies and internationally harmonised protocols, particularly for metadata reporting. Our recommendations aim to support both small-scale projects and large initiatives, directing collective efforts to facilitate efficient sampling, vouchering and sample processing for future genomic studies.

Subcellular fractionation of Euglena gracilis has been conducted for over 50 years in various forms by numerous research groups. The development of this technique is closely tied to the specific organelle or fraction required for specific purposes. In this chapter, we describe our approach to this process and discuss the insights we gain from it. Sucrose and iodixanol gradients are employed to separate the main organelles of interest; however, these methods alone do not lead to the complete purification of the organelles.

Hypotheses concerning eukaryogenesis, the evolution of eukaryotic cells, differ in the relative timing of mitochondrial acquisition. Recently, a serial endosymbiotic hypothesis proposed that hydrogenosomes and mitosomes (MROs) in Metamonada originated from an independent endosymbiosis, later replaced by Alphaproteobacteria-related mitochondria, contradicting the paradigm of mitochondrial presence in the last eukaryotic common ancestor. This serial endosymbiotic hypothesis implicitly predicts the scarcity of alphaproteobacterial genes from Metamonada genomes, because they never contained this endosymbiont. We tested this prediction using a set of 1,399 and 97 proteins inferred for the Metamonada ancestor and confined to their MROs, respectively. We detected five and 14 orthologous groups (OGs) with alphaproteobacterial affiliation in the respective datasets. None of these OGs was present in oxymonads, a Metamonada subgroup lacking MROs, thus serving as blank references. Our data are therefore consistent with the ruling paradigm that mitochondria and MROs originated from an Alphaproteobacterium during a single common endosymbiosis.

Gut protists are an important part of the microbial community that is often overlooked or limited to certain "flagship" species, mainly parasites. This also applies to Eurasian beavers (Castor fiber), which act as aquatic engineers in riparian habitats and have been identified as potential reservoirs of the well-known intestinal protist parasite - Giardia intestinalis. In this study, we focus on exploring protistan diversity in Eurasian beavers from freshly collected stool samples using molecular methods, including targeted barcoding of specific parasitic groups (Giardia, Cryptosporidium and Microsporidia) and general metabarcoding of the V4 region of 18S rRNA on the Illumina MiSeq platform. Two individuals were tested positive for Cryptosporidium spp. using the targeted barcoding. The metabarcoding approach revealed that the gut community was predominantly composed of gut commensals, Blastocystis, and trichomonads, found in every individual. More detailed investigation of Blastocystis confirmed the potential existence of a specific beaver subtype. Colpodellids and parasitic Eimeria spp. were only occasionally present. Geographic location of the host, unlike sex, has a significant impact on gut microbiota composition, though more research is needed due to limited sampling. Finally, we tested a new primer set for amplifying the V4 region of 18S rRNA, which shows promise for improving the detection of Metamonada protists in the gut samples.

Despite the growing interest in basic research on Euglena gracilis and its importance in bioproduction, several key questions regarding its cell biology remain unanswered. One of them concerns the mechanism of protein import into its secondary chloroplasts, which are derived from a green alga. In this study, we have proved the essentiality of 2 rhomboid pseudoproteases, DerL-1 and DerL-2, which are predicted to localize in the membrane of the chloroplast envelope. We demonstrate that these proteins belong to a euglenid-specific subgroup unrelated to other eukaryotic rhomboid pseudoproteases. Silencing these proteins using RNA interference leads to a distinct phenotype marked by culture bleaching and delayed growth. This is accompanied by the loss of typical chloroplast structure and a reduction in the expression of a substantial portion of the chloroplast proteome, especially proteins involved in photosynthesis. We hypothesize that these pseudoproteases are involved in chloroplast protein import by forming a core component of the translocon across the middle envelope membrane. This indicates a remarkable case of molecular convergence, as other eukaryotes utilize rhomboid pseudoproteases-unrelated to euglenid proteins-for protein import into plastids derived from red algae.

Expansion microscopy is a powerful technique, which increases the effective resolution of a fluorescence microscope by physically enlarging the specimen within a swellable polymer matrix. When combined with confocal microscopy, it enables affordable and easy-to-implement super-resolution imaging of protein localization in an entire cellular volume. Here, we describe in detail a protocol for the expansion of Euglenozoa, such as Trypanosoma brucei and Euglena gracilis, which achieves an average expansion factor of 4.6 in the case of T. brucei and 3.5 in the case of E. gracilis. This method is compatible with a broad range of available antibodies targeting endogenous proteins or small epitope tags. Additionally, we demonstrate the use of a fluorescent NHS ester for whole-proteome staining, BODIPY lipid stain, and the SYTOX nucleic acid stain for reproducible DNA labeling in expanded cells of these organisms. These reagents further increase the versatility of this simple yet robust super-resolution approach for studies of the cell biology of Euglenozoa.

Metamonada is a eukaryotic supergroup of free-living and parasitic anaerobic protists. Their characteristic feature is the presence of highly reduced mitochondria that have lost the ability to produce ATP by oxidative phosphorylation and in some cases even by substrate phosphorylation, with all ATP being imported from the cytosol. Given this striking difference in cellular ATP metabolism when compared to aerobic mitochondria, we studied the presence of mitochondrial carrier proteins (MCPs) mediating the transport of ATP across the inner mitochondrial membrane. Our bioinformatic analyses revealed remarkable reduction of MCP repertoire in Metamonada with striking loss of the major ADP/ATP carrier (AAC). Instead, nearly all species retained carriers orthologous to human SLC25A43 protein, a little-characterized MCP. Heterologous expression of metamonad SLC25A43 carriers confirmed their mitochondrial localization, and functional analysis revealed that SLC25A43 orthologues represent a distinct group of ATP transporters, which we designate as ATP-importing carriers (AIC). Together, our findings suggest that AIC facilitate the ATP import into highly reduced anaerobic mitochondria, compensating for their diminished or absent energy metabolism.

Monocercomonoides exilis is a model species of the amitochondrial eukaryotic group Oxymonadida, which makes it a suitable organism for studying the consequences of mitochondrial loss. Although M. exilis has an endobiotic lifestyle, it can be cultured in vitro in polyxenic conditions alongside an uncharacterized prokaryotic community, while attempts to create axenic cultures have not been successful. In this study, we used metagenomic sequencing, transcriptomics, and metabolomics to characterize the microbial consortium that supports the growth of M. exilis. We assembled genomes for 24 bacterial species and identified at least 30 species in total. M. exilis accounted for less than 1.5% of the DNA reads, while bacterial species dominated the sequence data and shifted in abundance over time. Our metabolic reconstruction and differential gene expression analyses show that the bacterial community relies on organic carbon oxidation, fermentation, and hydrogen production, but does not engage in methanogenesis. We observed rapid depletion of amino acids, nucleotides, glyceraldehyde, lactate, fatty acids, and alcohols in the medium, indicating a reliance on external nutrient recycling. The nitrogen cycle in this community is incomplete, with limited nitrogen fixation and no ammonia oxidation. Despite detailed metabolic profiling, we did not find any direct biochemical connections between M. exilis and the prokaryotes. Several bacterial species produce siderophores to assist themselves and others in the community in acquiring iron. However, M. exilis does not appear to benefit directly from siderophore-mediated iron transport and lacks known iron uptake pathways. This indicates that M. exilis may rely indirectly on the iron metabolism of other bacteria through phagocytosis. Additionally, some bacteria synthesize polyamines like spermidine and phosphatidylcholine, which M. exilis may need but cannot produce on its own. As the culture ages, M. exilis shows changes in gene expression consistent with starvation responses, including the upregulation of carbohydrate storage pathways and processes related to exocytosis. These findings provide new insights into microbial interactions within xenic cultures and emphasize the complex nature of maintaining amitochondriate eukaryotes in vitro.

This article explores the use of expansion microscopy, a technique that enhances resolution in fluorescence microscopy, on the autotrophic protist Euglena gracilis A modified protocol was developed to preserve the cell structures during fixation. Using antibodies against key cytoskeletal and organelle markers, α-tubulin, β-ATPase, and Rubisco activase, the microtubular structures, mitochondria, and chloroplasts were visualised. The organisation of the cytoskeleton corresponded to the findings from electron microscopy while allowing for the visualisation of the flagellar pocket in its entirety and revealing previously unnoticed details. This study offered insights into the shape and development of mitochondria and chloroplasts under varying conditions, such as culture ages and light cycles. This work demonstrated that expansion microscopy is a robust tool for visualising cellular structures in E. gracilis, an organism whose internal structures cannot be stained using standard immunofluorescence because of its complex pellicle. This technique also serves as a complement to electron microscopy, facilitating tomographic reconstructions in a routine fashion.

This article explores the use of expansion microscopy, a technique that enhances resolution in fluorescence microscopy, on the autotrophic protist Euglena gracilis . A modified protocol was developed to preserve the cell structures during fixation. Using antibodies against key cytoskeletal and organelle markers, α-tubulin, β-ATPase, and Rubisco activase, the microtubular structures, mitochondria, and chloroplasts were visualised. The organisation of the cytoskeleton corresponded to the findings from electron microscopy while allowing for the visualisation of the flagellar pocket in its entirety and revealing previously unnoticed details. This study offered insights into the shape and development of mitochondria and chloroplasts under varying conditions, such as culture ages and light cycles. This work demonstrated that expansion microscopy is a robust tool for visualising cellular structures in E. gracilis , an organism whose internal structures cannot be stained using standard immunofluorescence because of its complex pellicle. This technique also serves as a complement to electron microscopy, facilitating tomographic reconstructions in a routine fashion.

Molecular surveillance was widely used during the COVID-19 pandemic to detect rapidly emerging variants and monitor the transmission of SARS-CoV-2 within communities. In 2021, the Czech COVID-19 Genomics Consortium (COG-CZ) was set up to coordinate a new SARS-CoV-2 molecular surveillance network. In the Czech Republic, molecular surveillance employed whole genome sequencing (WGS) and variant discrimination polymerase chain reaction (VD-PCR) on samples collected through passive, active and sentinel surveillance. All WGS data was uploaded to GISAID and the PANGO lineages used by GISAID were compared to the main variants determined by VD-PCR. To assess the effectiveness and reliability of the gathered data in adapting pandemic responses, the capabilities and turnaround times of the molecular surveillance methods are evaluated. VD-PCR results were available within 48 h of sample collection for 81.5% of cases during the Delta/Omicron transition. WGS enabled the detection of low-frequency novel variants in infection clusters. WGS surveillance showed there was community spread of AY.20.1, a variant that gained novel mutations within the Czech Republic. Molecular surveillance informed the implementation of public health measures; temporal comparisons of restrictions and outcomes are described. Further areas for improvement have been identified for monitoring and managing future pandemics.

Euglena gracilis, a flagellated, single-celled organism shaped like a spindle, belongs to the Euglenozoa phylum. This protist is predominantly found in freshwater bodies such as lakes and ponds. Unique in its ability to perform photosynthesis and heterotrophic feeding, E. gracilis can produce organic materials on its own or absorb them from the environment. This intriguing blend of plant and animal traits, coupled with promising biotechnological applications, have made it a subject of extensive research. Notably, E. gracilis has been used for biofuel production and synthesis of various compounds such as amino acids and vitamins. For these applications, rapid assessment of single E. gracilis cells within populations is essential. This paper introduces recent advancements in high-throughput optical imaging technology that enables large-scale, single-cell analysis of E. gracilis. We review the benefits and limitations of various methods within this technology, along with discussing its future potential.

Understanding the origin of eukaryotic cells is one of the most difficult problems in all of biology. A key challenge relevant to the question of eukaryogenesis is reconstructing the gene repertoire of the last eukaryotic common ancestor (LECA). As data sets grow, sketching an accurate genomics-informed picture of early eukaryotic cellular complexity requires provision of analytical resources and a commitment to data sharing. Here, we summarise progress towards understanding the biology of LECA and outline a community approach to inferring its wider gene repertoire. Once assembled, a robust LECA gene set will be a useful tool for evaluating alternative hypotheses about the origin of eukaryotes and understanding the evolution of traits in all descendant lineages, with relevance in diverse fields such as cell biology, microbial ecology, biotechnology, agriculture, and medicine. In this Consensus View, we put forth the status quo and an agreed path forward to reconstruct LECA’s gene content.

Monocercomonoides exilis is the first known amitochondriate eukaryote. Loss of mitochondria in M. exilis ocurred after the replacement of the essential mitochondrial iron-sulfur cluster (ISC) assembly machinery by a unique, bacteria-derived, cytosolic SUF system. It has been hypothesized that the MeSuf pathway, in cooperation with proteins of the cytosolic iron-sulfur protein assembly (CIA) system, is responsible for the biogenesis of FeS clusters in M. exilis, yet biochemical evidence is pending. Here, we address the M. exilis MeSuf system and show that SUF genes, individually or in tandem, support the loading of iron-sulfur (FeS) clusters into the reporter protein IscR in Escherichia coli. The Suf proteins MeSufB, MeSufC, and MeSufDSU interact in vivo with one another and with Suf proteins of E. coli. In vitro, the M. exilis Suf proteins form large complexes of varying composition and hence may function as a dynamic biosynthetic system in the protist. The putative FeS cluster scaffold MeSufB-MeSufC (MeSufBC) forms multiple oligomeric complexes, some of which bind FeS clusters and form selectively only in the presence of adenosine nucleotides. The multi-domain fusion protein MeSufDSU binds a PLP cofactor and can form higher-order complexes with MeSufB and MeSufC. Our work demonstrates the biochemical property of M. exilis Suf proteins to act as a functional FeS cluster assembly system and provides insights into the molecular mechanism of this unique eukaryotic SUF system.

Protists are key players in the biosphere. Here, we provide a perspective on integrating protist culturing with omics approaches, imaging, and high-throughput single-cell manipulation strategies, concluding with actions required for a successful return of the golden age of protist culturing.

Short amplicon sequencing is a commonly used method to study the diversity of organisms in various habitats. The hypervariable regions of the small subunit rRNA gene (18S rDNA) are the most general barcodes for eukaryotes, which can provide detailed taxonomic information across a wide range of eukaryotic diversity. However, some organisms are often missed by universal primers, which have difficulty amplifying their barcodes. In this study, specific primers were designed for the amplification of the highly diverse 18S-V9 region of the Euglenozoa and Metamonada groups. The performance of the newly designed primers-V9Eug and V9Meta-was compared with the universal V9 primer on cultured communities derived from a range of freshwater environments of the Soos Natural Reserve and the Slavkov Forest in the Czech Republic. The V9Eug primer was more specific with Euglenozoa representing 91.8% of reads and 57.0% of OTUs, while the V9Meta primer showed lower specificity with only 48.4% of reads and 19.7% of OTUs assigned to Metamonada. Both the Euglenozoa and Metamonada primer pairs significantly improved recovery of their target groups compared to the universal V9 primer pair, detecting 2.7 and 1.8 times more OTUs, respectively. These results provide a more sensitive protocol for studying the diversity of these eukaryotic taxa.

Spatial arrangement of the cytoskeleton in the cells of protists has been used for decades for taxonomy and phylogenetic inference at various levels. In contrast, the protein composition of non-microtubular structures is mostly unknown. Exceptions are system I fibres in algae, which are built of striated fiber assemblins (SFA). Interestingly, SFAs are also components of a range of other, dissimilar structures, playing a role in the cortex of ciliates, cells division in apicomplexans, and adhesion of the parasite Giardia to the intestine. In a broad bioinformatic survey, we show the presence of three ancestral eukaryotic paralogues of SFA, and note that they are present in all ‗typical excavates‘ – simple flagellates bearing a ventral feeding groove. In one representative, Paratrimastix pyriformis, we localised one of the SFA paralogues using specific antibodies and expansion microscopy. We show that it co-localises specifically with structures attached to the basal body of the posterior flagellum, namely the right microtubular root, composite fibre, and B-fibre. As the morphology of the ‗typical excavates‘ may be ancestral to eukaryotes, we speculate that the role in the development or function of this feeding apparatus may be ancestral to the protein. ### Competing Interest Statement The authors have declared no competing interest.