Czech Academy of Sciences, Czech Republic
iDIPCON abstract
Diplonemids, which were found to be among the most abundant and diverse heterotrophic micro-eukaryotes in the world’s oceans, also known for a unique form of post-transcriptional processing in mitochondria, were until recently missing a type species that has hampered elucidation of their cell and molecular biology. In my talk, I am going to summarize the way how we managed to turn Diplonema papillatum (recently renamed Paradiplonema papillatum) into a model species. I will describe the path from our initial results when we showed stable but random integration of delivered extraneous DNA to later ones when we demonstrated that homologous recombination in P. papillatum. is functional and targeting various constructs to the required position in the nuclear genome is successful when 5' and 3' homologous regions longer than 1 kbp are used. Since this technique was established, we have been able to successfully label several dozens of proteins using Protein A or V5 tag. The proteins belong to both groups: 1/ with already known/expected functions and 2/ hypothetical ones. For both groups the tagging was used to determine their intracellular localization by immunofluorescence and identify their interacting partners that mainly for the second group should help to assign their function. To sum up, our collaborative work opened the door to functional studies in P. papillatum and making it another model system for study of marine micro-eukaryotes.
Dalhousie University, Canada
Czech Academy of Sciences, Czech Republic
iDIPCON abstract
Analysis of the global Tara Oceans V9 global metabarcoding dataset revealed that an
overlooked and minor lineage of heterotrophic protists, now termed eupelagonemids, is among the most
diverse and abundant planktonic eukaryotes in the sunlit oceans. This brought the eupelagonemids into
the focus of the scientific community as major players in the global ocean ecosystem, but also caused
a justified caution about interpretation and validation of these results. Especially because V4-based
metabarcoding datasets show almost no eupelagonemid signal. To date, no single strain is known from
culture, so most of the current knowledge about this elusive but important eukaryotic lineage is based
on the analysis of approximately 130bp-long amplicons of SSU rRNA and several very partial single-cell
genome drafts.
Nonetheless, they still provide a fairly solid picture of their ecology and diversity, showing that
eupelagonemids are abundant, cosmopolitan and make up most of the Discoban richness and biomass,
especially in the deeper oceans. The abundance, richness and community structure of eupelagonemids are
strongly dependent on geography, oxygen concentration, salinity and temperature, favouring tropical
and nutrient-rich regions while avoiding high oxygen concentration, high salinity and high algal
density. Detailed sampling along the depth and oxygen gradients in the Southern Ocean also shows a
strongly correlated distribution of syndinian parasitoids and eupelagonemids, suggesting their close
ecological association.
Finally, previous evidence suggested that the observed eupelagonemid richness may be inflated by
intragenomic variability of barcoding marker. To answer this question, we compared this parameter
in 19 eukaryotic phyla abundant in marine plankton and found its level to be comparable in all phyla.
Moreover, most of the variability observed at the barcode level could be attributed to methodological
errors and had no significant impact on relative eupelagonemid richness.
University of Tsukuba, Japan
Origins of family A DNA polymerases in Euglenozoa.
Mitochondria and plastids are the organelles derived from endosymbiotic bacteria in eukaryotic cells. Due to their bacterial origins, the two organelles retain their own genomes. Nevertheless, the machinery for maintaining organellar genomes has been intensively modified during eukaryotic evolution. A recent study surveying family A DNA polymerases (famA DNAPs) in phylogenetically broad eukaryotes revealed an unexpected complex evolution of famA DNAPs for DNA maintenance in organelles—different branches of the tree of eukaryotes established organellar DNAPs with distinct evolutionary backgrounds. Euglenozoa is a typical example of the eukaryotic lineage establishing an unique set of mitochondrion (mt)-localized famA DNAPs. Two types of Euglenozoa-specific, mt-localized famA DNAPs have been known: PolIA and PolIBCD+. An apparently mt-localized type of PolIBCD+ has been found so far in Kinetoplastea and Diplonemea, and the ancestral DNAP of PolIBCD+ appeared to be a DNA polymerase I (PolI) in an autographivirus. PolIA type was most likely established in the common ancestor of Euglenozoa, as this type of mt-localized DNAPs has been identified in Symbiontida, Euglenida, Diplonemea, and Kinetoplastea. The evolutionary affinity between PolIA and a nucleus-localized DNAP found in diverse eukaryotes, Polθ, has been known. Nevertheless, it has not been studied the evolutionary trajectory of how Euglenozoa-specific PolIA emerged from Polθ with a paneukaryotic distribution. To solve the uncertainty in the origin of PolIA, we first explored the phylogenetic diversity of Polθ and then searched for the Polθ sequences with the specific phylogenetic affinity to Euglenozoa-specific PolIA in this study.
University of British Columbia, Canada
iDIPCON abstract
Eupelagonemids, formerly known as Deep Sea Pelagic Diplonemids I (DSPD I), are among the most abundant and diverse heterotrophic protists in the deep ocean, but little else is known about their ecology, evolution, or biology in general. Originally recognized solely as a large clade of environmental ribosomal subunit RNA gene sequences (SSU rRNA), branching with a smaller sister group DSPD II, they were postulated to be diplonemids, a poorly-studied branch of Euglenozoa. Although new diplonemids have been cultivated and studied in depth in recent years, the lack of cultured eupelagonemids has limited data to a handful of light micrographs, partial SSU rRNA gene sequences, a small number of genes from single amplified genomes (SAGs), and only a single formal described species, Eupelagonema oceanica. To determine exactly where this clade goes in the tree of eukaryotes and begin to address the overall absence of biological information about this apparently ecologically important group, we recently conducted single-cell transcriptomics of two eupelagonemid cells. A SSU phylogeny shows these two cells represent distinct subclades within eupelagonemids, each different from E. oceanica. Phylogenomic analysis based on a 125-gene matrix contrasts with the findings based on ecological survey data, and shows eupelagonemids branch sister to the diplonemid subgroup Hemistasiidae. We recently collected and photodocumented 30 additional eupelagonemid cells for single-cell transcriptomics. In preliminary SSU phylogenies these cells are scattered across the tree of eupelagenomids, representing a broad sampling of eupelagonemid diversity.
University of California, Davis, United States
iDIPCON abstract
Mitochondrial Complex I is a crucial component of the electron transport chain, composed of multiple subunits encoded by both nuclear and mitochondrial genomes. In euglenozoans, the NDUFS1 subunit, which plays an essential role in the catalytic core, exhibits unusual patterns of fragmentation and gene transfer. Euglena has two nuclear-encoded subunits, NDUFS1A and NDUFS1B. In contrast, Diplonema species have only the NDUFS1A portion in the nucleus, while a divergent mitochondrial open reading frame (Y4) appears to functionally replace NDUFS1B. Structures reveal that Y4 maintains partial topological similarity to NDUFS1B despite significant sequence divergence and structural degeneration. Comparisons with kinetoplastids reveal no clear homolog of Y4 or NDUFS1B, suggesting extreme divergence or gene loss. These findings provide evidence for a dynamic evolutionary process involving gene fragmentation, nuclear gene transfer, RNA-level processing, and differential retention of mitochondrial genes. This study highlights the remarkable plasticity of the euglenozoan mitochondrial genome and offers new insights into the functional and structural adaptations underlying mitochondrial Complex I evolution.
CNRS & Université Paris-Saclay, France
CSIC - Spanish National Research Council, Spain
iDIPCON abstract
Small protists (up to 3 µm in size) are integral members of marine ecosystems in terms of cell abundance and biomass, and play crucial roles in food webs and biogeochemical cycles. Their diversity can hardly be investigated by standard microscopy, and instead a variety of molecular tools have been applied, unveiling a large phylogenetic diversity and the presence of novel lineages within the assemblage. The development of molecular tools have advanced in parallel with extensive temporal and spatial surveys in the marine realm. Here I will present data on the contribution of the different eukaryotic groups in a the global circumnavigation Malaspina cruise, and in a monthly sampling at the Mediterranean Sea spanning more than 20 years. The presence of diplonemids will be evaluated in particular, taken advantage of parallel metagenomic data. The combination of large marine datasets and new sequencing capabilities allows a detailed study of the temporal and spatial dynamics of protists in the ocean.
School of Biological Sciences, The University of Edinburgh, United Kingdom
Insights into diplonemid intermediary metabolism
Intermediary metabolism, i.e., the network of reactions that provides cells with metabolic
energy and biosynthetic intermediates, has been experimentally studied in great detail in Trypanosoma brucei and
to a lesser extent in some other trypanosomatids, but not in organisms belonging to other kinetoplastid taxa and
in diplonemids. However, analysis of genome sequences and using knowledge about the enzymes and network organization
of trypanosomatids, has also provided insight into how these latter organisms metabolize their nutrients to produce
ATP and intermediates for biosynthesis.
Published experimental studies of Diplonema papillatum revealed that it may preferentially use polysaccharides
as carbon and energy source but is also capable of metabolizing amino acids obtained from poly- and oligopeptides.
Indeed, genome analysis of different diplonemids predicted that they possess complete glycolytic and gluconeogenic
pathways, and a TCA cycle and respiratory system, by which the metabolism of these substrates can occur, as well as
other important pathways of intermediary metabolism such as the pentose-phosphate pathway and pathways for
β-oxidation and biosynthesis of fatty acids. Importantly, the overall cellular organization of the intermediary
metabolic network of diplonemids is similar to that in trypanosomatids, i.e., involves essentially three
compartments –cytosol, glycosomes and mitochondrion-– but also displays specific differences.
Glycosomes have previously been identified in trypanosomes as peroxisome-related organelles harbouring the major
part of the glycolytic/gluconeogenic pathway, and were subsequently detected in all kinetoplastids analyzed, and thus
more recently also in diplonemids. However, they are absent from the third subclade of the phylum Euglenozoa, the
euglenids, which contain peroxisomes without glycolytic enzymes.
We will discuss the similarities and differences of intermediary metabolism of diplonemids and trypanosomatids, with
emphasis on the metabolic compartmentalization. The peculiar properties of glycosomes and the compartmentalization of
enzymes and cofactors within these organelles have some important consequences for the metabolism and biology of these
organisms. In this respect, we will also mention some difficulties in deriving conclusions about metabolic patterns
based predicting subcellular location from genome analysis. In addition, we will address the possible evolutionary
driver(s) of the unique form of metabolic compartmentalization observed in diplonemids and kinetoplastids, also with
reference to some current ideas about the functions and evolution of peroxisomes in general.
Comenius University, Slovakia
Powering the protist: ATP production in Paradiplonema papillatum
1 Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia
2 Life Science Research Centre, Faculty of Science, University of Ostrava, Czech Republic
3 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech Republic
4 Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic
Paradiplonema papillatum is a free-living marine euglenozoan protist. The phylum Euglenozoa is a diverse group encompassing various families, including diplonemids, trypanosomatids, euglenids, and symbiontids, each characterized by distinct features. Intriguingly, the P. papillatum genome reveals a unique mosaic of characteristics borrowed from these diverse Euglenozoa lineages. Current data suggest a highly adaptable metabolism, potentially enabling survival across a broad range of environmental conditions. Indeed, P. papillatum demonstrates tolerance to significant variations in temperature and salinity, nutrient depletion, and even the presence of toxic pollutants. However, some aspects of its predicted metabolic potential appear to contradict experimental observations. For instance, despite possessing genes encoding anaerobic enzymes, P. papillatum does not appear to thrive in anaerobic environments; respiration seems crucial for cofactor oxidation and maintaining membrane potential. While its mitochondria appear typical of aerobic organisms, P. papillatum exhibits several unusual mitochondrial adaptations. These include a modified tricarboxylic acid cycle with an additional shunt, a unique pyruvate dehydrogenase complex composition, and a distinct electron transport chain. This chain incorporates both standard oxidative phosphorylation complexes and alternative enzymes.
University of Warsaw, Poland
3D reconstruction of a diplonemid: ultrastructure of cell division
1 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech Republic
2 Institute of Evolutionary Biology, Faculty of Biology, University of Warsaw, Poland
3 Faculty of Sciences, University of South Bohemia, České Budějovice, Czech Republic
Diplonemids are among the most species-rich and abundant eukaryotes in the World’s Oceans.
Over the past decade, multiple studies have focused on the diversity and ecology of diplonemids,
their metabolic capabilities as well as their complex mitochondrial and nuclear genomes. However,
until recently, our knowledge on fine structure of diplonemids was mostly restricted to the use of
classical transmission electron microscopy, providing limited information about their cellular
architecture. Detailed knowledge of the cell biology in this eukaryotic lineage is essential for
correct interpretation of ecological and molecular data.
We provide the first comprehensive study
of diplonemid cellular architecture based on whole-cell 3D reconstruction of Lacrimia vacuolata,
assembled from serial block-face scanning electron microscopy images, additionally complemented with
light, fluorescence, and transmission electron microscopy. Out of all euglenozoans, similar research
is available for only two model species – parasitic Trypanosoma brucei (kinetoplastids) and
photosynthetic Euglena gracilis (euglenids), which are poor representations for the bulk
diversity of free-living phagotrophic biflagellated euglenozoans. We illustrate the position, morphology,
and fine structure of the feeding and flagellar apparatuses and identify subcellular peculiarities
previously not reported in this clade. We further present the first ultrastructural description of
diplonemid cell division, including degradation and reassembly of complex feeding and flagellar
apparatuses as well as the partitioning of organelles into daughter cells. Finally, we have identified
a novel, ultrastructurally complex organelle termed ‘COLV’ (Center for Organization of Layered Vesicles).
We suggest that the colv is involved in food processing and membrane trafficking, and describe its
close association with other components of the cellular digestive system.
Université de Montréal, Canada
Proteomics of diplonemid mitochondrial complexes.
Omics approaches that focus on genomes and transcriptomes have become the foundation for studies of
living systems. But it is through proteomics that we can gain a more accurate view of inner workings
of cells and their dynamic nature, assign functions to unfamiliar genetic content or reveal how
individual components collaborate to carry out biological processes. Nowhere are these strategies more
important than in trying to understand idiosyncrasies of fast-evolving and quirky organisms, of which
diplonemids are a prime example.
While providing a more comprehensive overview, whole-cell proteomics is hampered—as in other
organisms—by the high dynamic range of protein concentrations in a diplonemid cell and struggles to
detect low-abundance proteins. To overcome current technical limitations, more focused approaches
using genetic engineering-mediated targeting of proteins of interest proved rewarding, bringing forth
new insights into vesicular transport, kinetochore architecture, respiratory chain complexes, as well
as mitochondrial ribosome biogenesis. In my presentation, I will focus on recent studies of
mitochondrial protein and ribonucleo-protein complexes to showcase how these divide-and-conquer
strategies are not only showing what makes diplonemids special but are also beginning to provide
first real clues how this happens.
Arizona State University, United States
Japan Agency for Marine-Earth Science and Technology, Japan
iDIPCON abstract
The first diplonemid species was described in 1913 and by 2017 only 10 more species had been described. However, their number has now increased to 22 at present and more diplonemids are waiting for formal description. This great acceleration of taxonomic studies on diplonemids is majorly facilitated by the development of Hemi-medium. By using Hemi-medium, many diplonemids have been established as clonal axenic cultures from water samples and subjected to advanced studies. In this talk, I would like to present the diplonemids that were established by utilizing Hemi-medium and their studies. As Hemi-medium is also useful for other protist species and the cultures of non-diplonemid protists have been also established up to now, the research carried out on them will also be presented.