Unique features of conventional and nonconventional introns in Euglena gracilis.

BACKGROUND: Nuclear introns in Euglenida have been understudied. This study aimed to investigate nuclear introns in Euglenida by identifying a large number of introns in Euglena gracilis (E. gracilis), including cis-spliced conventional and nonconventional introns, as well as trans-spliced outrons. We also examined the sequence characteristics of these introns. RESULTS: A total of 28,337 introns and 11,921 outrons were identified. Conventional and nonconventional introns have distinct splice site features; the former harbour canonical GT/C-AG splice sites, whereas the latter are capable of forming structured motifs with their terminal sequences. We observed that short introns had a preference for canonical GT-AG introns. Notably, conventional introns and outrons in E. gracilis exhibited a distinct cytidine-rich polypyrimidine tract, in contrast to the thymidine-rich tracts observed in other organisms. Furthermore, the SL-RNAs in E. gracilis, as well as in other trans-splicing species, can form a recently discovered motif called the extended U6/5' ss duplex with the respective U6s. We also describe a novel type of alternative splicing pattern in E. gracilis. The tandem repeat sequences of introns in this protist were determined, and their contents were comparable to those in humans. CONCLUSIONS: Our findings highlight the unique features of E. gracilis introns and provide insights into the splicing mechanism of these introns, as well as the genomics and evolution of Euglenida.

A chromosome-level genome assembly for the paramylon-producing microalga Euglena gracilis.

Euglena gracilis (E. gracilis), pivotal in the study of photosynthesis, endosymbiosis, and chloroplast development, is also an industrial microalga for paramylon production. Despite its importance, E. gracilis genome exploration faces challenges due to its intricate nature. In this study, we achieved a chromosome-level de novo assembly (2.37 Gb) using Illumina, PacBio, Bionano, and Hi-C data. The assembly exhibited a contig N50 of 619 Kb and scaffold N50 of 1.12 Mb, indicating superior continuity. Approximately 99.83% of the genome was anchored to 46 chromosomes, revealing structural insights. Repetitive elements constituted 58.84% of the sequences. Functional annotations were assigned to 39,362 proteins, enhancing interpretative power. BUSCO analysis confirmed assembly completeness at 80.39%. This first high-quality E. gracilis genome offers insights for genetics and genomics studies, overcoming previous limitations. The impact extends to academic and industrial research, providing a foundational resource.

Changes in Gravitaxis and Gene-Expression in an Euglena gracilis Culture over Time.

Age-dependent changes in the transcription levels of 5-day-old Euglena gracilis cells, which showed positive gravitaxis, 6-day-old cells without gravitactic orientation, and older cells (9- and 11-day-old, which displayed a precise negative gravitaxis) were determined through microarray analysis. Hierarchical clustering of four independent cell cultures revealed pronounced similarities in transcription levels at the same culture age, which proves the reproducibility of the cultivation method. Employing the non-oriented cells from the 6-day-old culture as a reference, about 2779 transcripts were found to be differentially expressed. While positively gravitactic cells (5-day-old culture) showed only minor differences in gene expression compared to the 6-day reference, pronounced changes of mRNAs (mainly an increase) were found in older cells compared to the reference culture. Among others, genes coding for adenylyl cyclases, photosynthesis, and metabolic enzymes were identified to be differentially expressed. The investigated cells were grown in batch cultures, so variations in transcription levels most likely account for factors such as nutrient depletion in the medium and self-shading. Based on these findings, a particular transcript (e.g., transcript 19556) was downregulated using the RNA interference technique. Gravitaxis and phototaxis were impaired in the transformants, indicating the role of this transcript in signal transduction. Results of the experiment are discussed regarding the increasing importance of E. gracilis in biotechnology as a source of valuable products and the possible application of E. gracilis in life-support systems.

High-efficiency genome editing by Cas12a ribonucleoprotein complex in Euglena gracilis.

Transgene-free genome editing based on clustered regularly interspaced short palindromic repeats (CRISPR) technology is key to achieving genetic engineering in microalgae for basic research and industrial applications. Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, cosmetics and biofuels. However, methods for the genetic manipulation of E. gracilis are still limited. Here, we developed a high-efficiency, transgene-free genome editing method for E. gracilis using Lachnospiraceae bacterium CRISPR-associated protein 12a (LbCas12a) ribonucleoprotein (RNP) complex, which complements the previously established Cas9 RNP-based method. Through the direct delivery of LbCas12a-containing RNPs, our method reached mutagenesis rates of approximately 77.2-94.5% at two different E. gracilis target genes, Glucan synthase-like 2 (EgGSL2) and a phytoene synthase gene (EgcrtB). Moreover, in addition to targeted mutagenesis, we demonstrated efficient knock-in and base editing at the target site using LbCas12a-based RNPs with a single-stranded DNA donor template in E. gracilis. This study extends the genetic engineering capabilities of Euglena to accelerate its basic use for research and engineering for bioproduction.

Ras superfamily GTPases and signal transduction in Euglena gracilis.

Biological complexity is challenging to define, but can be considered through one or more features, including overall genome size, number of genes, morphological features, multicellularity, number of life cycle stages and the ability to adapt to different environments. Euglena gracilis meets several of these criteria, with a large genome of ∼38,000 protein coding genes and a considerable ability to survive under many different conditions, some of which can be described as challenging or harsh. Potential molecular exemplars of complexity tying these aspects together are signalling pathways, including GTPases, kinases and ubiquitylation, which increase the functionality of the gene-encoded proteome manyfold. Each of these examples can modulate both protein activity and gene expression. To address the connection between genome size and complexity I have undertaken a brief, and somewhat qualitative, survey of the small ras-like GTPase superfamily of E. gracilis. Unexpectedly, apart from Rab-GTPases which control intracellular transport and organelle identify, the size of the GTPase cohort is modest, and, for example, has not scaled with gene number when compared to the close relatives, trypanosomatids. I suggest that understanding the functions of this protein family will be vital to uncovering the complexity of E. gracilis biology.

Circular extrachromosomal DNA in Euglena gracilis under normal and stress conditions.

Extrachromosomal circular DNA (eccDNA) enhances genomic plasticity, augmenting its coding and regulatory potential. Advances in high-throughput sequencing have enabled the investigation of these structural variants. Although eccDNAs have been investigated in numerous taxa, they remained understudied in euglenids. Therefore, we examined eccDNAs predicted from Illumina sequencing data of Euglena gracilis Z SAG 1224-5/25, grown under optimal photoperiod and exposed to UV irradiation. We identified approximately 1000 unique eccDNA candidates, about 20% of which were shared across conditions. We also observed a significant enrichment of mitochondrially encoded eccDNA in the UV-irradiated sample. Furthermore, we found that the heterogeneity of eccDNA was reduced in UV-exposed samples compared to cells that were grown in optimal conditions. Hence, eccDNA appears to play a role in the response to oxidative stress in Euglena, as it does in other studied organisms. In addition to contributing to the understanding of Euglena genomes, our results contribute to the validation of bioinformatics pipelines on a large, non-model genome.

Effects of polystyrene microplastics on Euglena gracilis: Intracellular distribution and the protozoan transcriptional responses.

Microplastics (MPs) introduced into aquatic environments inevitably interact with aquatic organisms such as plankton, potentially yielding adverse effects on the aquatic ecosystem. The extent to which MPs can infiltrate planktonic cells and evoke a molecular response remains largely unknown. In the present study, the internalization of fluorescently labeled polystyrene (PS) MPs on Euglena gracilis cells was investigated, determining the transcriptional responses within protozoa after an 8-day exposure period. The results showed that exposure to 25 mg/L PS-MPs for 8 days, significantly inhibited protozoan growth (P < 0.05) and decreased the chlorophyll a content of E. gracilis. The photosynthetic efficiency of E. gracilis was suppressed by MPs after 4 days, and then recovered to control values by the eighth day. Fluorescence imaging confirmed the presence of MPs in E. gracilis. Transcriptomic analysis revealed the influence of PS-MPs on a diverse range of transcriptional processes, encompassing oxidative phosphorylation, oxidation-reduction process, photosynthesis, and antioxidant enzymes. Notably, a majority of the differentially expressed genes (DEGs) exhibited down-regulation. Furthermore, PS-MPs disturbed the transcriptional regulation of chloroplasts and photosynthesis. These findings indicate a direct interaction between PS-MPs and organelles within E. gracilis cells following internalization, thereby disrupting regular gene expression patterns and posing a substantial environmental risk to the aquatic ecosystem.

Optimal conditions of algal breeding using neutral beam and applying it to breed Euglena gracilis strains with improved lipid accumulation.

Microalgae are considered to be more useful and effective to use in biomass production than other photosynthesis organisms. However, microalgae need to be altered to acquire more desirable traits for the relevant purpose. Although neutron radiation is known to induce DNA mutations, there have been few studies on its application to microalgae, and the optimal relationship between irradiation intensity and mutation occurrence has not been established. In this study, using the unicellular red alga Cyanidioschyzon merolae as a model, we analyzed the relationship between the absorbed dose of two types of neutrons, high-energy (above 1 MeV) and thermal (around 25 meV) neutrons, and mutation occurrence while monitoring mutations in URA5.3 gene encoding UMP synthase. As a result, the highest mutational occurrence was observed when the cells were irradiated with 20 Gy of high-energy neutrons and 13 Gy of thermal neutrons. Using these optimal neutron irradiation conditions, we next attempted to improve the lipid accumulation of Euglena gracilis, which is a candidate strain for biofuel feedstock production. As a result, we obtained several strains with a maximum 1.3-fold increase in lipid accumulation compared with the wild-type. These results indicate that microalgae breeding by neutron irradiation is effective.

The impact of elevated sulfur and nitrogen levels on cadmium tolerance in Euglena species.

Heavy metal (HM) pollution threatens human and ecosystem health. Current methods for remediating water contaminated with HMs are expensive and have limited effect. Therefore, bioremediation is being investigated as an environmentally and economically viable alternative. Freshwater protists Euglena gracilis and Euglena mutabilis were investigated for their tolerance to cadmium (Cd). A greater increase in cell numbers under Cd stress was noted for E. mutabilis but only E. gracilis showed an increase in Cd tolerance following pre-treatment with elevated concentrations of S or N. To gain insight regarding the nature of the increased tolerance RNA-sequencing was carried out on E. gracilis. This revealed transcript level changes among pretreated cells, and additional differences among cells exposed to CdCl2. Gene ontology (GO) enrichment analysis reflected changes in S and N metabolism, transmembrane transport, stress response, and physiological processes related to metal binding. Identifying these changes enhances our understanding of how these organisms adapt to HM polluted environments and allows us to target development of future pre-treatments to enhance the use of E. gracilis in bioremediation relating to heavy metals.

Unveiling Immunomodulatory Effects of Euglena gracilis in Immunosuppressed Mice: Transcriptome and Pathway Analysis.

The immunomodulatory effects of Euglena gracilis (Euglena) and its bioactive component, ß-1,3-glucan (paramylon), have been clarified through various studies. However, the detailed mechanisms of the immune regulation remain to be elucidated. This study was designed not only to investigate the immunomodulatory effects but also to determine the genetic mechanisms of Euglena and ß-glucan in cyclophosphamide (CCP)-induced immunosuppressed mice. The animals were orally administered saline, Euglena (800 mg/kg B.W.) or ß-glucan (400 mg/kg B.W.) for 19 days, and CCP (80 mg/kg B.W.) was subsequently administered to induce immunosuppression in the mice. The mice exhibited significant decreases in body weight, organ weight, and the spleen index. However, there were significant improvements in the spleen weight and the spleen index in CCP-induced mice after the oral administration of Euglena and ß-glucan. Transcriptome analysis of the splenocytes revealed immune-related differentially expressed genes (DEGs) regulated in the Euglena- and ß-glucantreated groups. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that pathways related with interleukin (IL)-17 and cAMP play significant roles in regulating T cells, B cells, and inflammatory cytokines. Additionally, Ptgs2, a major inflammatory factor, was exclusively expressed in the Euglena-treated group, suggesting that Euglena's beneficial components, such as carotenoids, could regulate these genes by influencing immune lymphocytes and inflammatory cytokines in CCP-induced mice. This study validated the immunomodulatory effects of Euglena and highlighted its underlying mechanisms, suggesting a positive contribution to the determination of phenotypes associated with immune-related diseases and the research and development of immunotherapies.

Biochemical and Functional Profiling of Thioredoxin-Dependent Cytosolic GPX-like Proteins in Euglena gracilis.

Unlike plants and animals, the phytoflagellate Euglena gracilis lacks catalase and contains a non-selenocysteine glutathione peroxidase-like protein (EgGPXL), two peroxiredoxins (EgPrx1 and EgPrx4), and one ascorbate peroxidase in the cytosol to maintain reactive oxygen species (ROS) homeostasis. In the present study, the full-length cDNA of three cytosolic EgGPXLs was obtained and further characterized biochemically and functionally. These EgGPXLs used thioredoxin instead of glutathione as an electron donor to reduce the levels of H2O2 and t-BOOH. The specific peroxidase activities of these enzymes for H2O2 and t-BOOH were 1.3 to 4.9 and 0.79 to 3.5 µmol/min/mg protein, respectively. Cytosolic EgGPXLs and EgPrx1/EgPrx4 were silenced simultaneously to investigate the synergistic effects of these genes on the physiological function of E. gracilis. The suppression of cytosolic EgGPXL genes was unable to induce any critical phenomena in Euglena under normal (100 µmol photons m-2 s-1) and high-light conditions (350 µmol photons m-2 s-1) at both autotrophic and heterotrophic states. Unexpectedly, the suppression of EgGPXL genes was able to rescue the EgPrx1/EgPrx4-silenced cell line from a critical situation. This study explored the potential resilience of Euglena to ROS, even with restriction of the cytosolic antioxidant system, indicating the involvement of some compensatory mechanisms.