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

The trypanosomatid flagellates possess in their single mitochondrion a highly complex kinetoplast (k)DNA, which is composed of interlocked circular molecules of two types. Dozens of maxicircles represent a classical mitochondrial genome, and thousands of minicircles encode guide (g)RNAs, which direct the processive and essential uridine insertion/deletion messenger RNA (mRNA) editing of maxicircle transcripts. While the details of kDNA structure and this type of RNA editing are well established, our knowledge mostly relies on a narrow foray of intensely studied human parasites of the genera Leishmania and Trypanosoma. Here, we analyzed kDNA, its expression, and RNA editing of two members of the poorly characterized genus Vickermania with very different cultivation histories. In both Vickermania species, the gRNA-containing heterogeneous large (HL)-circles are atypically large with multiple gRNAs each. Examination of Vickermania spadyakhi HL-circle loci revealed a massive redundancy of gRNAs relative to the editing needs. In comparison, the HL-circle repertoire of extensively cultivated Vickermania ingenoplastis is greatly reduced. It correlates with V. ingenoplastis-specific loss of productive editing of transcripts encoding subunits of respiratory chain complex I and corresponding lack of complex I activity. This loss in a parasite already lacking genes for subunits of complexes III and IV suggests an apparent requirement for its mitochondrial adenosine triphosphate (ATP) synthase to work in reverse to maintain membrane potential. In contrast, V. spadyakhi retains a functional complex I that allows ATP synthase to work in its standard direction.

• Keywords
• ATP synthase, RNA editing, Vickermania, kinetoplast DNA, trypanosomatids,
• MeSH
• RNA Editing * genetics   MeSH
• Genome, Mitochondrial MeSH
• Genome, Protozoan * MeSH
• DNA, Kinetoplast * genetics   MeSH
• Evolution, Molecular * MeSH
• Trypanosomatina * genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Kinetoplast * MeSH

The exon junction complex (EJC) is a key player in metazoan mRNA quality control and is placed upstream of the exon-exon junction after splicing. Its inner core is composed of Magoh, Y14, eIF4AIII and BTZ and the outer core of proteins involved in mRNA splicing (CWC22), export (Yra1), translation (PYM) and nonsense mediated decay (NMD, UPF1/2/3). Trypanosoma brucei encodes only two genes with introns, but all mRNAs are processed by trans-splicing. The presence of three core EJC proteins and a potential BTZ homologue (Rbp25) in trypanosomes has been suggested to adapt of the EJC function to mark trans-spliced mRNAs. We analysed trypanosome EJC components and noticed major differences between eIF4AIII and Magoh/Y14: (i) whilst eIF4AIII is essential, knocking out both Magoh and Y14 elicits only a mild growth phenotype (ii) eIF4AIII localization is mostly nucleolar, while Magoh and Y14 are nucleolar and nucleoplasmic but excluded from the cytoplasm (iii) eIF4AIII associates with nucleolar proteins and the splicing factor CWC22, but not with Y14 or Magoh, while Magoh and Y14 associate with each other, but not with eIF4AIII, CWC22 or nucleolar proteins. Our data argue against the presence of a functional EJC in trypanosomes, but indicate that eIF4AIII adopted non-EJC related, essential functions, while Magoh and Y14 became redundant. Trypanosomes also possess homologues to the NMD proteins UPF1 and UPF2. Depletion of UPF1 causes only a minor reduction in growth and phylogenetic analyses show several independent losses of UPF1 and UPF2, as well as complete loss of UPF3 in the Kinetoplastida group, indicating that UPF1-dependent NMD is not essential. Regardless, we demonstrate that UPF1 depletion restores the mRNA levels of a PTC reporter. Altogether, we show that the almost intron-less trypanosomes are in the process of losing the canonical EJC/NMD pathways: Y14 and Magoh have become redundant and the still-functional UPF1-dependent NMD pathway is not essential.

• MeSH
• Eukaryotic Initiation Factor-4A metabolism   genetics   MeSH
• Exons genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Nonsense Mediated mRNA Decay * MeSH
• Protozoan Proteins * metabolism   genetics   MeSH
• RNA Helicases * metabolism   genetics   MeSH
• RNA Splicing MeSH
• Trypanosoma brucei brucei * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Eukaryotic Initiation Factor-4A MeSH
• RNA, Messenger MeSH
• Protozoan Proteins * MeSH
• RNA Helicases * MeSH

Lotmaria passim is a ubiquitous trypanosomatid parasite of honey bees nestled within the medically important subfamily Leishmaniinae. Although this parasite is associated with honey bee colony losses, the original draft genome-which was completed before its differentiation from the closely related Crithidia mellificae-has remained the reference for this species despite lacking improvements from newer methodologies. Here, we report the updated sequencing, assembly, and annotation of the BRL-type (Bee Research Laboratory) strain (ATCC PRA-422) of Lotmaria passim. The nuclear genome assembly has been resolved into 31 complete chromosomes and is paired with an assembled kinetoplast genome consisting of a maxicircle and 30 minicircle sequences. The assembly spans 33.7 Mb and contains very little repetitive content, from which our annotation of both the nuclear assembly and kinetoplast predicted 10,288 protein-coding genes. Analyses of the assembly revealed evidence of a recent chromosomal duplication event within chromosomes 5 and 6 and provided evidence for a high level of aneuploidy in this species, mirroring the genomic flexibility employed by other trypanosomatids as a means of adaptation to different environments. This high-quality reference can therefore provide insights into adaptations of trypanosomatids to the thermally regulated, acidic, and phytochemically rich honey bee hindgut niche, which offers parallels to the challenges faced by other Leishmaniinae during the challenges they undergo within insect vectors, during infection of mammals, and exposure to antiparasitic drugs throughout their multi-host life cycles. This reference will also facilitate investigations of strain-specific genomic polymorphisms, their role in pathogenicity, and the development of treatments for pollinator infection.

• Keywords
• Lotmaria passim strain BRL, ATCC PRA-422, Hi-C, Leishmaniinae, PacBio, Trypanosomatidae, aneuploidy, monoxenous, polyploidy, trypanosomatid,
• MeSH
• Molecular Sequence Annotation MeSH
• Phylogeny MeSH
• Genome, Protozoan MeSH
• Genomics methods   MeSH
• Evolution, Molecular * MeSH
• Trypanosomatina * genetics   classification   MeSH
• Bees parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Leishmania is a genus of the family Trypanosomatidae that unites obligatory parasitic flagellates causing a variety of vector-borne diseases collectively called leishmaniasis. The symptoms range from relatively innocuous skin lesions to complete failures of visceral organs. The disease is exacerbated if a parasite harbors Leishmania RNA viruses (LRVs) of the family Pseudototiviridae. Screening a novel isolate of L. braziliensis, we revealed that it possesses not a toti-, but a bunyavirus of the family Leishbuviridae. To the best of our knowledge, this is a very first discovery of a bunyavirus infecting a representative of the Leishmania subgenus Viannia. We suggest that these viruses may serve as potential factors of virulence in American leishmaniasis and encourage researchers to test leishmanial strains for the presence of not only LRVs, but also other RNA viruses.

• MeSH
• Bunyaviridae classification   genetics   isolation & purification   MeSH
• Phylogeny MeSH
• Leishmania braziliensis * genetics   isolation & purification   MeSH
• Humans MeSH
• Orthobunyavirus genetics   classification   isolation & purification   physiology   MeSH
• RNA Viruses genetics   classification   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Trypanosomatids are obligate parasites of animals, predominantly insects and vertebrates, and flowering plants. Monoxenous species, representing the vast majority of trypanosomatid diversity, develop in a single host, whereas dixenous species cycle between two hosts, of which primarily insect serves as a vector. To explore in-depth the diversity of insect trypanosomatids including their co-infections, sequence profiling of their 18S rRNA gene was used for true bugs (Hemiptera; 18% infection rate) and flies (Diptera; 10%) in Cuba. Out of 48 species (molecular operational taxonomic units) belonging to the genera Vickermania (16 spp.), Blastocrithidia (7), Obscuromonas (4), Phytomonas (5), Leptomonas/Crithidia (5), Herpetomonas (5), Wallacemonas (2), Kentomonas (1), Angomonas (1) and two unnamed genera (1 + 1), 38 species have been encountered for the first time. The detected Wallacemonas and Angomonas species constitute the most basal lineages of their respective genera, while Vickermania emerged as the most diverse group. The finding of Leptomonas seymouri, which is known to rarely infect humans, confirms that Dysdercus bugs are its natural hosts. A clear association of Phytomonas with the heteropteran family Pentatomidae hints at its narrow host association with the insect rather than plant hosts. With a focus on multiple infections of a single fly host, using deep Nanopore sequencing of 18S rRNA, we have identified co-infections with up to 8 trypanosomatid species. The fly midgut was usually occupied by several Vickermania species, while Herpetomonas and/or Kentomonas species prevailed in the hindgut. Metabarcoding was instrumental for analysing extensive co-infections and also allowed the identification of trypanosomatid lineages and genera.

• Keywords
• biodiversity, diptera, heteroptera, host specificity, monoxenous trypanosomatids, multiple infections, nanopore sequencing, phylogeny, systematics,
• MeSH
• Diptera genetics   MeSH
• Phylogeny * MeSH
• Hemiptera parasitology   genetics   MeSH
• Coinfection * parasitology   MeSH
• DNA, Protozoan genetics   analysis   MeSH
• RNA, Ribosomal, 18S * genetics   analysis   MeSH
• Trypanosomatina * genetics   classification   isolation & purification   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Cuba epidemiology   MeSH
• Names of Substances
• DNA, Protozoan MeSH
• RNA, Ribosomal, 18S * MeSH

BACKGROUND: Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. RESULTS: We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. CONCLUSIONS: In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.

• Keywords
• Blastocrithidia, In silico, Metabolic predictions, Non-canonical genetic code, Trypanosomatid,
• MeSH
• Eukaryota genetics   MeSH
• Genetic Code MeSH
• Parasites * genetics   MeSH
• Codon, Terminator MeSH
• Trypanosoma brucei brucei * genetics   MeSH
• Trypanosomatina * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Codon, Terminator MeSH

RNA viruses play an important role in Leishmania biology and virulence. Their presence was documented in three (out of four) Leishmania subgenera. Sauroleishmania of reptiles remained the only underinvestigated group. In this work, we analyzed the viral occurrence in Sauroleishmania spp. and detected RNA viruses in three out of seven isolates under study. These viruses were of two families-Narnaviridae and Totiviridae. Phylogenetic inferences demonstrated that totiviruses from L. adleri and L. tarentolae group together within a larger cluster of LRV2s, while a narnavirus of L. gymnodactyli appeared as a phylogenetic relative of narnaviruses of Blechomonas spp. Taken together, our work not only expanded the range of trypanosomatids that can host RNA viruses but also shed new light on the evolution and potential routes of viral transmission in these flagellates.

• Keywords
• L. (S.) gymnodactyli, L. (S.) hoogstraali, L. (S.) tarentolae, LRV2, Leishmania (Sauroleishmania) adleri, Narnaviridae,
• MeSH
• Phylogeny MeSH
• Leishmania * MeSH
• Humans MeSH
• Reptiles MeSH
• RNA Viruses * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Protists of the family Trypanosomatidae (phylum Euglenozoa) have gained notoriety as parasites affecting humans, domestic animals, and agricultural plants. However, the true extent of the group's diversity spreads far beyond the medically and veterinary relevant species. We address several knowledge gaps in trypanosomatid research by undertaking sequencing, assembly, and analysis of genomes from previously overlooked representatives of this protistan group. RESULTS: We assembled genomes for twenty-one trypanosomatid species, with a primary focus on insect parasites and Trypanosoma spp. parasitizing non-human hosts. The assemblies exhibit sizes consistent with previously sequenced trypanosomatid genomes, ranging from approximately 18 Mb for Obscuromonas modryi to 35 Mb for Crithidia brevicula and Zelonia costaricensis. Despite being the smallest, the genome of O. modryi has the highest content of repetitive elements, contributing nearly half of its total size. Conversely, the highest proportion of unique DNA is found in the genomes of Wallacemonas spp., with repeats accounting for less than 8% of the assembly length. The majority of examined species exhibit varying degrees of aneuploidy, with trisomy being the most frequently observed condition after disomy. CONCLUSIONS: The genome of Obscuromonas modryi represents a very unusual, if not unique, example of evolution driven by two antidromous forces: i) increasing dependence on the host leading to genomic shrinkage and ii) expansion of repeats causing genome enlargement. The observed variation in somy within and between trypanosomatid genera suggests that these flagellates are largely predisposed to aneuploidy and, apparently, exploit it to gain a fitness advantage. High heterogeneity in the genome size, repeat content, and variation in chromosome copy numbers in the newly-sequenced species highlight the remarkable genome plasticity exhibited by trypanosomatid flagellates. These new genome assemblies are a robust foundation for future research on the genetic basis of life cycle changes and adaptation to different hosts in the family Trypanosomatidae.

• Keywords
• Dixenous, Genome assembly, Monoxenous, Parasite, Protist, Trypanosomatids, Whole-genome sequencing,
• MeSH
• Acclimatization MeSH
• Aneuploidy MeSH
• Genome Size MeSH
• Trypanosomatina * genetics   MeSH
• Agriculture MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH