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

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

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

Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread. One of the key life cycle stages is the haptomonad form, which attaches to insect tissues through its flagellum. This adhesion, conserved across kinetoplastid parasites, is implicated in having an important function within their life cycles and hence in disease transmission. Here, we discover the kinetoplastid-insect adhesion proteins (KIAPs), which localise in the attached Leishmania flagellum. Deletion of these KIAPs impairs cell adhesion in vitro and prevents Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. Additionally, loss of parasite adhesion in the sand fly results in reduced physiological changes to the fly, with no observable damage of the stomodeal valve and reduced midgut swelling. These results provide important insights into a comprehensive understanding of the Leishmania life cycle, which will be critical for developing transmission-blocking strategies.

• MeSH
• Cell Adhesion MeSH
• Flagella * metabolism   MeSH
• Insect Vectors parasitology   MeSH
• Insect Proteins metabolism   genetics   MeSH
• Host-Parasite Interactions MeSH
• Leishmania * physiology   genetics   metabolism   MeSH
• Leishmaniasis parasitology   transmission   MeSH
• Protozoan Proteins metabolism   genetics   MeSH
• Psychodidae * parasitology   MeSH
• Life Cycle Stages MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Insect Proteins MeSH
• Protozoan Proteins 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.

• Keywords
• Blastocrithidia, Mitoviridae, Narnaviridae, Obscuromonas, Qin-like virus, dsRNA viruses,
• 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

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.

• Keywords
• Krebs cycle, NAD+, NADP+, TCA cycle, cofactor preference, isocitrate dehydrogenase,
• MeSH
• Isocitrate Dehydrogenase * genetics   metabolism   MeSH
• Isocitrates metabolism   MeSH
• NAD * metabolism   MeSH
• NADP metabolism   MeSH
• Protein Isoforms MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Isocitrate Dehydrogenase * MeSH
• Isocitrates MeSH
• isocitric acid MeSH Browser
• NAD * MeSH
• NADP MeSH
• Protein Isoforms MeSH

In this work, we investigated parasites of the firebug Pyrrhocoris apterus in Austria and demonstrated that in addition to the extensively studied Leptomonas pyrrhocoris, it can also be infected by Blastocrithidia sp. and by a mermithid, which for the first time has been characterized using molecular methods. This diversity can be explained by the gregarious lifestyle, as well as the coprophagous and cannibalistic behavior of the insect hosts that makes them susceptible to various parasites. In addition, we showed no tight association of the L. pyrrhocoris haplotypes and geographical locations (at least, considering the relatively small scale of locations in Austria) implying that the natural populations of L. pyrrhocoris are mixed due to the mobility of their firebug hosts.

• Keywords
• Blastocrithidia, Firebugs, Mermithidae, Pyrrhocoris apterus,
• MeSH
• Heteroptera * parasitology   MeSH
• Parasites * MeSH
• Trypanosomatina * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Austria MeSH

BACKGROUND: Trypanosomatids are parasitic flagellates well known because of some representatives infecting humans, domestic animals, and cultural plants. Many trypanosomatid species bear RNA viruses, which, in the case of human pathogens Leishmania spp., influence the course of the disease. One of the close relatives of leishmaniae, Leptomonas pyrrhocoris, has been previously shown to harbor viruses of the groups not documented in other trypanosomatids. At the same time, this species has a worldwide distribution and high prevalence in the natural populations of its cosmopolitan firebug host. It therefore represents an attractive model to study the diversity of RNA viruses. RESULTS: We surveyed 106 axenic cultures of L. pyrrhocoris and found that 64 (60%) of these displayed 2-12 double-stranded RNA fragments. The analysis of next-generation sequencing data revealed four viral groups with seven species, of which up to five were simultaneously detected in a single trypanosomatid isolate. Only two of these species, a tombus-like virus and an Ostravirus, were earlier documented in L. pyrrhocoris. In addition, there were four new species of Leishbuviridae, the family encompassing trypanosomatid-specific viruses, and a new species of Qinviridae, the family previously known only from metatranscriptomes of invertebrates. Currently, this is the only qinvirus with an unambiguously determined host. Our phylogenetic inferences suggest reassortment in the tombus-like virus owing to the interaction of different trypanosomatid strains. Two of the new Leishbuviridae members branch early on the phylogenetic tree of this family and display intermediate stages of genomic segment reduction between insect Phenuiviridae and crown Leishbuviridae. CONCLUSIONS: The unprecedented wide range of viruses in one protist species and the simultaneous presence of up to five viral species in a single Leptomonas pyrrhocoris isolate indicate the uniqueness of this flagellate. This is likely determined by the peculiarity of its firebug host, a highly abundant cosmopolitan species with several habits ensuring wide distribution and profuseness of L. pyrrhocoris, as well as its exposure to a wider spectrum of viruses compared to other trypanosomatids combined with a limited ability to transmit these viruses to its relatives. Thus, L. pyrrhocoris represents a suitable model to study the adoption of new viruses and their relationships with a protist host.

• Keywords
• Leishbuviridae, Ostravirus, Pyrrhocoris apterus, Qinviridae, Tombus-like viruses,
• MeSH
• Phylogeny MeSH
• Animals, Domestic MeSH
• Humans MeSH
• RNA Viruses * genetics   MeSH
• Trypanosomatina * genetics   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Monoxenous (insect-restricted) trypanosomatids are highly diverse and abundant in nature. While many papers focus on the taxonomy and distribution of these parasites, studies on their biology are still scarce. In particular, this concerns trypanosomatids inhabiting the ubiquitous mosquitoes. To shed light on the circulation of monoxenous trypanosomatids with the participation of mosquitoes, we performed a multifaceted study combining the examination of naturally- and experimentally-infected insects using light and electron microscopy and molecular identification of parasites. Our examination of overwintering mosquitoes (genera Culex and Culiseta) revealed that their guts contained living trypanosomatids, which can be spread during the next season. Experimental infections with Crithidia spp. demonstrated that imagines represent permissive hosts, while larvae are resistant to these parasites. We argue that for the parasites with wide specificity, mosquitoes act as facultative hosts. Other trypanosomatids may have specific adaptations for vertical transmission in these insects at the expense of their potential to infect a wider range of hosts and, consequently, abundance in nature.

• Keywords
• Crithidia, Paratrypanosoma, Trypanosomatidae, experimental infection, facultative host, overwintering mosquitoes, prevalence, specificity, transmission,
• Publication type
• Journal Article MeSH