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.

• Klíčová slova
• Lotmaria passim strain BRL, ATCC PRA-422, Hi-C, Leishmaniinae, PacBio, Trypanosomatidae, aneuploidy, monoxenous, polyploidy, trypanosomatid,
• MeSH
• anotace sekvence MeSH
• fylogeneze MeSH
• genom protozoální MeSH
• genomika metody   MeSH
• molekulární evoluce * MeSH
• Trypanosomatina * genetika   klasifikace   MeSH
• včely parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Introns, Kinetoplastea, Poly(A) polymerase, RNA helicase, RNA-binding protein, Splicing, Trypanosomatidae,
• MeSH
• fylogeneze MeSH
• introny * genetika   MeSH
• Kinetoplastida genetika   MeSH
• molekulární evoluce MeSH
• protozoální geny genetika   MeSH
• protozoální proteiny genetika   MeSH
• trans-splicing * genetika   MeSH
• Trypanosomatina genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protozoální proteiny 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 klasifikace   genetika   izolace a purifikace   MeSH
• fylogeneze MeSH
• Leishmania braziliensis * genetika   izolace a purifikace   MeSH
• lidé MeSH
• Orthobunyavirus genetika   klasifikace   izolace a purifikace   fyziologie   MeSH
• RNA-viry genetika   klasifikace   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Trypanosomatids (Euglenozoa) are a diverse group of unicellular flagellates predominately infecting insects (monoxenous species) or circulating between insects and vertebrates or plants (dixenous species). Monoxenous trypanosomatids harbor a wide range of RNA viruses belonging to the families Narnaviridae, Totiviridae, Qinviridae, Leishbuviridae, and a putative group of tombus-like viruses. Here, we focus on the subfamily Blastocrithidiinae, a previously unexplored divergent group of monoxenous trypanosomatids comprising two related genera: Obscuromonas and Blastocrithidia. Members of the genus Blastocrithidia employ a unique genetic code, in which all three stop codons are repurposed to encode amino acids, with TAA also used to terminate translation. Obscuromonas isolates studied here bear viruses of three families: Narnaviridae, Qinviridae, and Mitoviridae. The latter viral group is documented in trypanosomatid flagellates for the first time. While other known mitoviruses replicate in the mitochondria, those of trypanosomatids appear to reside in the cytoplasm. Although no RNA viruses were detected in Blastocrithidia spp., we identified an endogenous viral element in the genome of B. triatomae indicating its past encounter(s) with tombus-like viruses.

• Klíčová slova
• Blastocrithidia, Mitoviridae, Narnaviridae, Obscuromonas, Qin-like virus, dsRNA viruses,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• biodiversity, diptera, heteroptera, host specificity, monoxenous trypanosomatids, multiple infections, nanopore sequencing, phylogeny, systematics,
• MeSH
• Diptera genetika   MeSH
• fylogeneze * MeSH
• Hemiptera parazitologie   genetika   MeSH
• koinfekce * parazitologie   MeSH
• protozoální DNA genetika   analýza   MeSH
• RNA ribozomální 18S * genetika   analýza   MeSH
• Trypanosomatina * genetika   klasifikace   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Kuba epidemiologie   MeSH
• Názvy látek
• protozoální DNA MeSH
• RNA ribozomální 18S * MeSH

The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.

• MeSH
• buněčné jádro * genetika   metabolismus   MeSH
• editace RNA * MeSH
• genetický kód MeSH
• genom mitochondriální * MeSH
• guide RNA, Kinetoplastida genetika   metabolismus   MeSH
• kodon genetika   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• otevřené čtecí rámce genetika   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• RNA transferová * genetika   metabolismus   MeSH
• terminační kodon genetika   MeSH
• Trypanosomatina genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• guide RNA, Kinetoplastida MeSH
• kodon MeSH
• messenger RNA MeSH
• protozoální proteiny MeSH
• RNA transferová * MeSH
• terminační kodon MeSH

In Uzbekistan, the number of reported leishmaniasis cases is rising at the alarming rate. In this work, we studied the phlebotomine sand fly (Diptera: Phlebotominae) diversity in the foci of cutaneous leishmaniasis in the Surxondaryo Region of Uzbekistan and compared it with the data obtained for the same area 50 years ago, when infection prevalence was reportedly low. We found that the implicated vector for zoonotic leishmaniasis, P. papatasi, remained eudominant; the proportion of implicated anthroponotic leishmaniasis vector, P. sergenti, rose significantly from averaged 5.4 to 41.4%; Phlebotomus alexandri, a suspected visceral leishmaniasis vector, was eudominant at two sites, and a second suspected vector for this disease, P. longiductus, was newly recorded in the region. We conclude that the increase in the documented cases of cutaneous leishmaniasis in the Surxondaryo Region of Uzbekistan may be connected to the changes in fauna of sand flies vectoring Leishmania spp.

• Klíčová slova
• Diversity, Leishmania, Phlebotomus,
• MeSH
• hmyz - vektory MeSH
• leishmanióza kožní * epidemiologie   MeSH
• leishmanióza viscerální * epidemiologie   MeSH
• Phlebotomus * MeSH
• Psychodidae * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Uzbekistán epidemiologie   MeSH

Isocitrate dehydrogenase is an enzyme converting isocitrate to α-ketoglutarate in the canonical tricarboxylic acid (TCA) cycle. There are three different types of isocitrate dehydrogenase documented in eukaryotes. Our study points out the complex evolutionary history of isocitrate dehydrogenases across kinetoplastids, where the common ancestor of Trypanosomatidae and Bodonidae was equipped with two isoforms of the isocitrate dehydrogenase enzyme: the NADP+-dependent isocitrate dehydrogenase 1 with possibly dual localization in the cytosol and mitochondrion and NADP+-dependent mitochondrial isocitrate dehydrogenase 2. In the extant trypanosomatids, isocitrate dehydrogenase 1 is present only in a few species suggesting that it was lost upon separation of Trypanosoma spp. and replaced by the mainly NADP+-dependent cytosolic isocitrate dehydrogenase 3 of bacterial origin in all the derived lineages. In this study, we experimentally demonstrate that the omnipresent isocitrate dehydrogenase 2 has a dual localization in both mitochondrion and cytosol in at least four species that possess only this isoform. The apparent lack of the NAD+-dependent isocitrate dehydrogenase activity in trypanosomatid mitochondrion provides further support to the existence of the noncanonical TCA cycle across trypanosomatids and the bidirectional activity of isocitrate dehydrogenase 3 when operating with NADP+ cofactor instead of NAD+. This observation can be extended to all 17 species analyzed in this study, except for Leishmania mexicana, which showed only low isocitrate dehydrogenase activity in the cytosol. The variability in isocitrate oxidation capacity among species may reflect the distinct metabolic strategies and needs for reduced cofactors in particular environments.

• Klíčová slova
• Krebs cycle, NAD+, NADP+, TCA cycle, cofactor preference, isocitrate dehydrogenase,
• MeSH
• isocitrátdehydrogenasa * genetika   metabolismus   MeSH
• isocitráty metabolismus   MeSH
• NAD * metabolismus   MeSH
• NADP metabolismus   MeSH
• protein - isoformy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• isocitrátdehydrogenasa * MeSH
• isocitráty MeSH
• isocitric acid MeSH Prohlížeč
• NAD * MeSH
• NADP MeSH
• protein - isoformy 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.

• Klíčová slova
• Blastocrithidia, In silico, Metabolic predictions, Non-canonical genetic code, Trypanosomatid,
• MeSH
• Eukaryota genetika   MeSH
• genetický kód MeSH
• paraziti * genetika   MeSH
• terminační kodon MeSH
• Trypanosoma brucei brucei * genetika   MeSH
• Trypanosomatina * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• terminační kodon MeSH