Culicoides (Diptera: Ceratopogonidae) biting midges are a diverse group of insect vectors that transmit pathogens affecting humans, livestock, and wild animals. Among them, Oropouche virus, African Horse sickness virus, and bluetongue virus are the most notable pathogens. However, comparatively little is known about which Culicoides species serve as vectors of wildlife parasites affecting wild birds globally, including the malaria-like parasite of the genus Haemoproteus (Haemosporida: Haemoproteidae) and kinetoplastid Trypanosoma (Trypanosomatida: Trypanosomatidae). Beyond the direct impact of their bites, infections by these parasites negatively affect wild birds from early developmental stages, significantly influencing their ecology and evolution. Here, we present a comprehensive review of the role of Culicoides species in the transmission of these two genera of avian parasites in Europe: Haemoproteus and Trypanosoma. We identify key information and methods used to study Culicoides-bird-parasite interactions, from insect sampling to vector competence assessment. Additionally, we highlight key knowledge gaps and propose future research directions in this area.

• Keywords
• Culicoides, Haemoproteus, Trypanosoma, Avian malaria, Birds, Blood parasites, Vectors,
• MeSH
• Ceratopogonidae * parasitology   physiology   MeSH
• Animals, Wild parasitology   MeSH
• Haemosporida physiology   isolation & purification   MeSH
• Insect Vectors * parasitology   MeSH
• Bird Diseases * transmission   parasitology   epidemiology   MeSH
• Protozoan Infections, Animal * transmission   parasitology   epidemiology   MeSH
• Birds * parasitology   MeSH
• Trypanosoma physiology   isolation & purification   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Geographicals
• Europe epidemiology   MeSH

Diplonemids are highly diverse and abundant marine plankton with significant ecological importance. However, little is known about their biology, even in the model diplonemid Paradiplonema papillatum whose genome sequence is available. Examining the subcellular localization of proteins using fluorescence microscopy is a powerful approach to infer their putative function. Here, we report a plasmid-based method that enables YFP-tagging of a gene at the endogenous locus. By examining the localization of proteins whose homologs are involved in chromosome organization or segregation in other eukaryotes, we discovered several notable features in mitotically dividing P. papillatum cells. Cohesin is enriched on condensed interphase chromatin. During mitosis, chromosomes organize into two rings (termed mitotic rings herein) that surround the elongating nucleolus and align on a bipolar spindle. Homologs of chromosomal passenger complex components (INCENP, two Aurora kinases and KIN-A), a CLK1 kinase, meiotic chromosome axis protein SYCP2L1, spindle checkpoint protein Mad1 and microtubule regulator XMAP215 localize in between the two mitotic rings. In contrast, a Mad2 homolog localizes near basal bodies as in trypanosomes. By representing the first molecular characterization of mitotic mechanisms in P. papillatum and raising many questions, this study forms the foundation for dissecting mitotic mechanisms in diplonemids.

• Keywords
• Euglenozoa, chromosome, diplonemid, kinetochore, kinetoplastid,
• MeSH
• Spindle Apparatus metabolism   MeSH
• Chromosomal Proteins, Non-Histone metabolism   MeSH
• Chromosomes metabolism   MeSH
• Dinoflagellida * genetics   metabolism   cytology   MeSH
• Mitosis * MeSH
• Cell Cycle Proteins metabolism   MeSH
• Chromosome Segregation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromosomal Proteins, Non-Histone MeSH
• Cell Cycle Proteins MeSH

In Europe, trypanosomes are not considered a serious threat to humans or domestic animals; however, their potential ecological impact on wildlife remains largely unknown. This study investigates presence of trypanosomes in European brown hare (Lepus europaeus) in Czechia, where this species is an important game species and plays a significant role in ecosystems. Using nested PCR targeting 18S rDNA, trypanosome DNA was detected in two of 435 blood samples, representing the first confirmation of trypanosomes in the genus Lepus. Subsequent sequencing identified Trypanosoma pestanai, a species previously reported in the European badger (Meles meles). These findings suggest that T. pestanai may have a broader host range than previously recognized, emphasizing the need for further research into its ecological impact and its potential transmission dynamics in wildlife populations.

• Keywords
• Blood parasite, European hare, Herpetosoma, Leporidae, Nested PCR, Wildlife,
• Publication type
• Journal Article MeSH

UNLABELLED: Trypanosomatids are among the most extensively studied protists due to their parasitic interactions with insects, vertebrates, and plants. Recently, Blastocrithidia nonstop was found to depart from the canonical genetic code, with all three stop codons reassigned to encode amino acids (UAR for glutamate and UGA for tryptophan), and UAA having dual meaning also as a termination signal (glutamate and stop). To explore features linked to this phenomenon, we analyzed the genomes of four Blastocrithidia and four Obscuromonas species, the latter representing a sister group employing the canonical genetic code. We found that all Blastocrithidia species encode cognate tRNAs for UAR codons, possess a distinct 4 bp anticodon stem tRNATrpCCA decoding UGA, and utilize UAA as the only stop codon. The distribution of in-frame reassigned codons is consistently non-random, suggesting a translational burden avoided in highly expressed genes. Frame-specific enrichment of UAA codons immediately following the genuine UAA stop codon, not observed in Obscuromonas, points to a specific mode of termination. All Blastocrithidia species possess specific mutations in eukaryotic release factor 1 and a unique acidic region following the prion-like N-terminus of eukaryotic release factor 3 that may be associated with stop codon readthrough. We infer that the common ancestor of the genus Blastocrithidia already exhibited a GC-poor genome with the non-canonical genetic code. Our comparative analysis highlights features associated with this extensive stop codon reassignment. This cascade of mutually dependent adaptations, driven by increasing AU-richness in transcripts and frequent emergence of in-frame stops, underscores the dynamic interplay between genome composition and genetic code plasticity to maintain vital functionality. IMPORTANCE: The genetic code, assigning amino acids to codons, is almost universal, yet an increasing number of its alterations keep emerging, mostly in organelles and unicellular eukaryotes. One such case is the trypanosomatid genus Blastocrithidia, where all three stop codons were reassigned to amino acids, with UAA also serving as a sole termination signal. We conducted a comparative analysis of four Blastocrithidia species, all with the same non-canonical genetic code, and their close relatives of the genus Obscuromonas, which retain the canonical code. This across-genome comparison allowed the identification of key traits associated with genetic code reassignment in Blastocrithidia. This work provides insight into the evolutionary steps, facilitating an extensive departure from the canonical genetic code that occurred independently in several eukaryotic lineages.

• Keywords
• AT-rich genomes, eukaryotic release factors, nuclear genetic code, reassigned codon, tRNA structure, termination of translation,
• MeSH
• Cell Nucleus * genetics   MeSH
• Phylogeny MeSH
• Genetic Code * MeSH
• Genome, Protozoan * MeSH
• Genomics MeSH
• Evolution, Molecular MeSH
• RNA, Transfer genetics   MeSH
• Codon, Terminator genetics   MeSH
• Trypanosomatina * genetics   classification   MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• RNA, Transfer MeSH
• Codon, Terminator MeSH

Blastocrithidia nonstop is a protist with a highly unusual nuclear genetic code, in which all three standard stop codons are reassigned to encode amino acids, with UAA also serving as a sole termination codon. In this study, we demonstrate that this parasitic flagellate is amenable to genetic manipulation, enabling gene ablation and protein tagging. Using preassembled Cas9 ribonucleoprotein complexes, we successfully disrupted and tagged the non-essential gene encoding catalase. These advances establish this single-celled eukaryote as a model organism for investigating the malleability and evolution of the genetic code in eukaryotes.

• Keywords
• CRISPR‐Cas9, codon reassignment, genetic code, model organism, trypanosomatids,
• MeSH
• Genetic Code * genetics   MeSH
• Catalase genetics   MeSH
• Protozoan Proteins genetics   MeSH
• Codon, Terminator genetics   MeSH
• Trypanosomatina * genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Catalase MeSH
• Protozoan Proteins MeSH
• Codon, Terminator MeSH

We present a genome assembly of the diplonemid Rhynchopus euleeides (Euglenozoa; Diplonemea; Diplonemea; Diplonemidae). The genome sequence is 199.0 megabases long, with most of the assembly scaffolded into 88 chromosomal pseudomolecules. The multipartite mitochondrial genome and the 2.0 megabase genome of Ca. Syngnamydia salmonis, a bacterial endosymbiont of R. euleeides, were also sequenced and assembled.

• Keywords
• Diplonemea, Euglenozoa, Rhynchopus euleeides, bacterial endosymbionts, chromosomal, genome sequence,
• Publication type
• Journal Article MeSH

Transposable elements (TEs) have the ability to move and amplify inside the host genome, making them a pivotal source of genome plasticity. Presently, only 4 TE clades (all classified as Class I retrotransposons) have been identified in trypanosomatids. We predicted repeat content and manually curated TEs across the genomes of 57 trypanosomatids, shedding light on their proportions, diversity and dynamics. Our analysis yielded 214 TE consensus sequence models across the dataset, with abundance ranging from 0.1% to 7.2%. We found evidence of recent transposon activity in most species, with notable bursts in the Vickermania, Lafontella, Porcisia and Angomonas spp., along with Leishmania (Mundinia) chancei, L. (M.) orientalis and L. (M.) procaviensis. We confirmed that the 4 TE clades have colonized virtually all lineages of trypanosomatids, potentially playing a role in shaping their genome architecture. The effort of this work culminated in the establishment of the Trypanosomatid TE Database 1.0, a resource designed to standardize the TE annotation process that can serve as a foundation for future studies on trypanosomatid TEs.

• Keywords
• CRE, INGI, SLACS, TATE, VIPER, mobilome, transposable elements, trypanosomatids,
• MeSH
• Phylogeny MeSH
• Genetic Variation * MeSH
• Genome, Protozoan * MeSH
• Evolution, Molecular * MeSH
• Retroelements genetics   MeSH
• DNA Transposable Elements * genetics   MeSH
• Trypanosomatina * genetics   classification   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Retroelements MeSH
• DNA Transposable Elements * MeSH

Avian trypanosomes (Trypanosoma, Kinetoplastea) are successful blood parasites occurring worldwide. These parasites are usually non-pathogenic to their avian hosts, thus neglected in studies regarding their life cycles and vectors. Several families of blood-sucking dipteran insects, including mosquitoes, have been identified as vectors of avian trypanosomes. Mosquitoes have been experimentally confirmed as vectors of Trypanosoma culicavium and Trypanosoma thomasbancrofti. In this study, we describe a third species of avian trypanosomes occurring in mosquitoes, designated as Trypanosoma tertium n. sp. This species can be distinguished from related trypanosome species based on morphology and small subunit rRNA gene sequence. Two isolates of T. tertium n. sp. obtained from a mosquito and a bird host were able to infect two subspecies of laboratory Culex pipiens mosquitoes, with infection rates reaching 60% and heavy infections in 90% of positive females. In infected mosquitoes, trypanosomes occurred as long epimastigotes in the midgut and short epimastigotes and rosettes in the hindgut. Putative infectious stages were detected in the diuretic liquid of infected mosquitoes, suggesting, besides transmission through ingestion of the infected vector, a possible transconjunctival infection. Among wild mosquitoes, avian trypanosomes were detected exclusively in Cx. pipiens with 3.3% total prevalence, while T. tertium n. sp. prevalence was only 0.08% among 1128 dissected Cx. pipiens individuals. In birds, T. tertium n. sp. was detected in 8 species within which the prevalence was 1.3% (686 birds), while it was 0.3% in total (3084 birds). We discuss the relationship of the newly described T. tertium n. sp. with other mosquito-transmitted trypanosomes.

• Keywords
• Culex, avian blood parasite, monoxenous Kinetoplastea, morphology, phylogeny, transmission, vector,
• MeSH
• Culex * parasitology   MeSH
• Phylogeny MeSH
• Mosquito Vectors * parasitology   MeSH
• Bird Diseases * parasitology   epidemiology   transmission   MeSH
• Prevalence MeSH
• Trypanosoma * genetics   isolation & purification   classification   growth & development   MeSH
• Trypanosomiasis * veterinary   parasitology   epidemiology   transmission   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

UNLABELLED: Transmission of genetic material from one generation to the next is a fundamental feature of all living cells. In eukaryotes, a macromolecular complex called the kinetochore plays crucial roles during chromosome segregation by linking chromosomes to spindle microtubules. Little is known about this process in evolutionarily diverse protists. Within the supergroup Discoba, Euglenozoa forms a speciose group of unicellular flagellates-kinetoplastids, euglenids, and diplonemids. Kinetoplastids have an unconventional kinetochore system, while euglenids have subunits that are conserved among most eukaryotes. For diplonemids, a group of extremely diverse and abundant marine flagellates, it remains unclear what kind of kinetochores are present. Here, we employed deep homology detection protocols using profile-versus-profile Hidden Markov Model searches and AlphaFold-based structural comparisons to detect homologies that might have been previously missed. Interestingly, we still could not detect orthologs for most of the kinetoplastid or canonical kinetochore subunits with few exceptions including a putative centromere-specific histone H3 variant (cenH3/CENP-A), the spindle checkpoint protein Mad2, the chromosomal passenger complex members Aurora and INCENP, and broadly conserved proteins like CLK kinase and the meiotic synaptonemal complex proteins SYCP2/3 that also function at kinetoplastid kinetochores. We examined the localization of five candidate kinetochore-associated proteins in the model diplonemid, Paradiplonema papillatum. PpCENP-A shows discrete dots in the nucleus, implying that it is likely a kinetochore component. PpMad2, PpCLKKKT10/19, PpSYCP2L1KKT17/18, and PpINCENP reside in the nucleus, but no clear kinetochore localization was observed. Altogether, these results point to the possibility that diplonemids evolved a hitherto unknown type of kinetochore system. IMPORTANCE: A macromolecular assembly called the kinetochore is essential for the segregation of genetic material during eukaryotic cell division. Therefore, characterization of kinetochores across species is essential for understanding the mechanisms involved in this key process across the eukaryotic tree of life. In particular, little is known about kinetochores in divergent protists such as Euglenozoa, a group of unicellular flagellates that includes kinetoplastids, euglenids, and diplonemids, the latter being a highly diverse and abundant component of marine plankton. While kinetoplastids have an unconventional kinetochore system and euglenids have a canonical one similar to traditional model eukaryotes, preliminary searches detected neither unconventional nor canonical kinetochore components in diplonemids. Here, we employed state-of-the-art deep homology detection protocols but still could not detect orthologs for the bulk of kinetoplastid-specific nor canonical kinetochore proteins in diplonemids except for a putative centromere-specific histone H3 variant. Our results suggest that diplonemids evolved kinetochores that do not resemble previously known ones.

• Keywords
• Diplonemea, Kinetoplastea, Paradiplonema, cell division, cenH3/CENP-A, kinetochore,
• MeSH
• Euglenozoa * genetics   metabolism   MeSH
• Phylogeny MeSH
• Kinetochores * metabolism   MeSH
• Protozoan Proteins metabolism   genetics   MeSH
• Chromosome Segregation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Protozoan Proteins MeSH

BACKGROUND: In trypanosomatids, a group of unicellular eukaryotes that includes numerous important human parasites, cis-splicing has been previously reported for only two genes: a poly(A) polymerase and an RNA helicase. Conversely, trans-splicing, which involves the attachment of a spliced leader sequence, is observed for nearly every protein-coding transcript. So far, our understanding of splicing in this protistan group has stemmed from the analysis of only a few medically relevant species. In this study, we used an extensive dataset encompassing all described trypanosomatid genera to investigate the distribution of intron-containing genes and the evolution of splice sites. RESULTS: We identified a new conserved intron-containing gene encoding an RNA-binding protein that is universally present in Kinetoplastea. We show that Perkinsela sp., a kinetoplastid endosymbiont of Amoebozoa, represents the first eukaryote completely devoid of cis-splicing, yet still preserving trans-splicing. We also provided evidence for reverse transcriptase-mediated intron loss in Kinetoplastea, extensive conservation of 5' splice sites, and the presence of non-coding RNAs within a subset of retained trypanosomatid introns. CONCLUSIONS: All three intron-containing genes identified in Kinetoplastea encode RNA-interacting proteins, with a potential to fine-tune the expression of multiple genes, thus challenging the perception of cis-splicing in these protists as a mere evolutionary relic. We suggest that there is a selective pressure to retain cis-splicing in trypanosomatids and that this is likely associated with overall control of mRNA processing. Our study provides new insights into the evolution of introns and, consequently, the regulation of gene expression in eukaryotes.

• Keywords
• Introns, Kinetoplastea, Poly(A) polymerase, RNA helicase, RNA-binding protein, Splicing, Trypanosomatidae,
• MeSH
• Phylogeny MeSH
• Introns * genetics   MeSH
• Kinetoplastida genetics   MeSH
• Evolution, Molecular MeSH
• Genes, Protozoan genetics   MeSH
• Protozoan Proteins genetics   MeSH
• Trans-Splicing * genetics   MeSH
• Trypanosomatina genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Protozoan Proteins MeSH