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1.
Mem Inst Oswaldo Cruz ; 115: e200504, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32578684

RESUMO

BACKGROUND: Biodiversity screens and phylogenetic studies are dependent on reliable DNA sequences in public databases. Biological collections possess vouchered specimens with a traceable history. Therefore, DNA sequencing of samples available at institutional collections can greatly contribute to taxonomy, and studies on evolution and biodiversity. METHODS: We sequenced part of the glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and the SSU rRNA (V7/V8) genes from 102 trypanosomatid cultures, which are available on request at www.colprot.fiocruz.br. OBJECTIVE: The main objective of this work was to use phylogenetic inferences, using the obtained DNA sequences and those from representatives of all Trypanosomatidae genera, to generate phylogenetic trees that can simplify new isolates screenings. FINDINGS: A DNA sequence is provided for the first time for several isolates, the phylogenetic analysis allowed the classification or reclassification of several specimens, identification of candidates for new genera and species, as well as the taxonomic validation of several deposits. MAIN CONCLUSIONS: This survey aimed at presenting a list of validated species and their associated DNA sequences combined with a short historical overview of each isolate, which can support taxonomic and biodiversity research and promote culture collections.


Assuntos
Biodiversidade , Código de Barras de DNA Taxonômico , Trypanosomatina/classificação , Trypanosomatina/genética , Filogenia
2.
Folia Parasitol (Praha) ; 672020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32350156

RESUMO

Here we describe the new trypanosomatid, Phytomonas borealis sp. n., from the midgut of the spiked shieldbugs, Picromerus bidens (Linnaeus), collected in two locations, Novgorod and Pskov Oblasts of Russia. The phylogenetic analyses, based on the 18S rRNA gene, demonstrated that this flagellate is a sister species to the secondary monoxenous Phytomonas nordicus Frolov et Malysheva, 1993, which was concurrently documented in the same host species in Pskov Oblast. Unlike P. nordicus, which can complete its development (including exit to haemolymph and penetration into salivary glands) in Picromerus bidens, the new species did not form any extraintestinal stages in the host. It also did not produce endomastigotes, indispensable for transmission in other Phytomonas spp. These observations, along with the fact that P. bidens overwinters at the egg stage, led us to the conclusion that the examined infections with P. borealis were non-specific. Strikingly, the flagellates from the Novgorod population contained prokaryotic endosymbionts, whereas the parasites from the second locality were endosymbiont-free. This is a first case documenting presence of intracellular symbiotic bacteria in Phytomonas spp. We suggest that this novel endosymbiotic association arose very recently and did not become obligate yet. Further investigation of P. borealis and its intracellular bacteria may shed light on the origin and early evolution of endosymbiosis in trypanosomatids.


Assuntos
Fenômenos Fisiológicos Bacterianos , Heterópteros/parasitologia , Simbiose , Trypanosomatina/classificação , Animais , Heterópteros/crescimento & desenvolvimento , Ninfa/crescimento & desenvolvimento , Ninfa/parasitologia , Filogenia , RNA de Protozoário/análise , RNA Ribossômico 18S/análise , Federação Russa , Trypanosomatina/microbiologia
3.
PLoS One ; 15(3): e0229536, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32155171

RESUMO

In this study, the presence of Leishmania DNA and blood feeding sources in phlebotomine sand fly species commonly present in Sicily were investigated. A total of 1,866 female sand flies including 176 blood fed specimens were sampled over two seasons in five selected sites in Sicily (southern Italy). Sergentomyia minuta (n = 1,264) and Phlebotomus perniciousus (n = 594) were the most abundant species at all the sites, while three other species from the genus Phlebotomus (i.e., P. sergenti n = 4, P. perfiliewi n = 3 and P. neglectus n = 1) were only sporadically captured. Twenty-eight out of the 1,866 (1.5%) sand flies tested positive for Leishmania spp. Leishmania tarentolae DNA was identified in 26 specimens of S. minuta, while the DNA of Leishmania donovani complex was detected in a single specimen each of S. minuta and P. perniciosus. Interestingly, seven S. minuta specimens (0.4%) tested positive for reptilian Trypanosoma sp. Blood sources were successfully identified in 108 out of 176 blood fed females. Twenty-seven out of 82 blood sources identified in fed females of P. perniciosus were represented by blood of wild rabbit, S. minuta mainly fed on humans (16/25), while the sole P. sergenti fed specimen took a blood meal on rat. Other vertebrate hosts including horse, goat, pig, dog, chicken, cow, cat and donkey were recognized as blood sources for P. perniciosus and S. minuta, and, surprisingly, no reptilian blood was identified in blood-fed S. minuta specimens. Results of this study agree with the well-known role of P. perniciosus as vector of L. infantum in the western Mediterranean; also, vector feeding preferences herein described support the hypothesis on the involvement of lagomorphs as sylvatic reservoirs of Leishmania. The detection of L. donovani complex in S. minuta, together with the anthropophilic feeding-behaviour herein observed, warrants further research to clarify the capacity of this species in the transmission of pathogens to humans and other animals.


Assuntos
Psychodidae/genética , Psychodidae/patogenicidade , Animais , DNA/genética , Comportamento Alimentar , Insetos Vetores , Leishmania/genética , Psychodidae/classificação , Estações do Ano , Sicília , Especificidade da Espécie , Trypanosomatina/genética
4.
PLoS One ; 15(1): e0227832, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945116

RESUMO

Here we characterized the development of the trypanosomatid Blastocrithidia raabei in the dock bug Coreus marginatus using light and electron microscopy. This parasite has been previously reported to occur in the host hemolymph, which is rather typical for dixenous trypanosomatids transmitted to a plant or vertebrate with insect's saliva. In addition, C. marginatus has an unusual organization of the intestine, which makes it refractory to microbial infections: two impassable segments isolate the anterior midgut portion responsible for digestion and absorption from the posterior one containing symbiotic bacteria. Our results refuted the possibility of hemolymph infection, but revealed that the refractory nature of the host provokes very aggressive behavior of the parasite and makes its life cycle more complex, reminiscent of that in some dixenous trypanosomatids. In the pre-barrier midgut portion, the epimastigotes of B. raabei attach to the epithelium and multiply similarly to regular insect trypanosomatids. However, when facing the impassable constricted region, the parasites rampage and either fiercely break through the isolating segments or attack the intestinal epithelium in front of the barrier. The cells of the latter group pass to the basal lamina and accumulate there, causing degradation of the epitheliocytes and thus helping the epimastigotes of the former group to advance posteriorly. In the symbiont-containing post-barrier midgut segment, the parasites either attach to bacterial cells and produce cyst-like amastigotes (CLAs) or infect enterocytes. In the rectum, all epimastigotes attach either to the cuticular lining or to each other and form CLAs. We argue that in addition to the specialized life cycle B. raabei possesses functional cell enhancements important either for the successful passage through the intestinal barriers (enlarged rostrum and well-developed Golgi complex) or as food reserves (vacuoles in the posterior end).


Assuntos
Infecções por Euglenozoa/veterinária , Heterópteros/imunologia , Interações Hospedeiro-Parasita/fisiologia , Estágios do Ciclo de Vida/fisiologia , Trypanosomatina/crescimento & desenvolvimento , Animais , Resistência à Doença , Infecções por Euglenozoa/imunologia , Infecções por Euglenozoa/parasitologia , Hemolinfa/parasitologia , Heterópteros/parasitologia , Mucosa Intestinal/diagnóstico por imagem , Mucosa Intestinal/parasitologia , Mucosa Intestinal/ultraestrutura , Microscopia Eletrônica , Trypanosomatina/patogenicidade , Trypanosomatina/ultraestrutura
5.
Parasitol Int ; 75: 102047, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31887394

RESUMO

Leishmania donovani, a protozoan parasite of family Trypanosomatidae, causes fatal visceral leishmaniasis (VL) in the Indian subcontinent and Africa and cutaneous leishmaniasis (CL) in Sri Lanka. Another member of Trypanosomatidae, Leptomonas seymouri, resembling Leishmania was discovered recently to co-exist with L. donovani in the clinical samples from India and Sri Lanka and therefore, interfere with its investigations. We earlier described a method for selective elimination of such co-existing L. seymouri from clinical samples of VL exploiting the differential growth of the parasites at 37 °C in vitro. Here, we explored ways for a rapid discriminatory diagnosis using high resolution melting (HRM) curves to detect co-occurring L. seymouri with L. donovani in clinical samples. Initial attempt with kDNA-minicircle (mitochondrial DNA) based HRM did not display different Tm values between L. donovani and L. seymouri. Surprisingly, all of their minicircle sequences co-existed in similar clades in the dendrogram analysis, although the kDNA sequences are known for its species and strain specific variations among the Trypanosomatids. However, an HRM analysis that targets the HSP70 gene successfully recognized the presence of L. seymouri in the clinical isolates. This discovery will facilitate rapid diagnosis of L. seymouri and further investigations in to this elusive organism, including the clinico-pathological implications of its co-existence with L. donovani in patients.


Assuntos
Coinfecção/diagnóstico , Infecções por Euglenozoa/diagnóstico , Leishmania donovani/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Trypanosomatina/isolamento & purificação , DNA de Cinetoplasto/análise
6.
Protist ; 170(6): 125698, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31760169

RESUMO

Strigomonas culicis is a kinetoplastid parasite of insects that maintains a mutualistic association with an intracellular symbiotic bacterium, which is highly integrated into the protist metabolism: it furnishes essential compounds and divides in synchrony with the eukaryotic nucleus. The protist, conversely, can be cured of the endosymbiont, producing an aposymbiotic cell line, which presents a diminished ability to colonize the insect host. This obligatory association can represent an intermediate step of the evolution towards the formation of an organelle, therefore representing an interesting model to understand the symbiogenesis theory. Here, we used shotgun proteomics to compare the S. culicis endosymbiont-containing and aposymbiotic strains, revealing a total of 11,305 peptides, and up to 2,213 proteins (2,029 and 1,452 for wild type and aposymbiotic, respectively). Gene ontology associated to comparative analysis between both strains revealed that the biological processes most affected by the elimination of the symbiont were the amino acid synthesis, as well as protein synthesis and folding. This large-scale comparison of the protein expression in S. culicis marks a step forward in the comprehension of the role of endosymbiotic bacteria in monoxenous trypanosomatid biology, particularly because trypanosomatids expression is mostly post-transcriptionally regulated.


Assuntos
Fenômenos Fisiológicos Bacterianos , Proteoma/genética , Simbiose/fisiologia , Trypanosomatina/microbiologia , Trypanosomatina/genética
7.
mBio ; 10(6)2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31772053

RESUMO

Current understanding of flagellum/cilium length regulation focuses on a few model organisms with flagella of uniform length. Leptomonas pyrrhocoris is a monoxenous trypanosomatid parasite of firebugs. When cultivated in vitro, L. pyrrhocoris duplicates every 4.2 ± 0.2 h, representing the shortest doubling time reported for trypanosomatids so far. Each L. pyrrhocoris cell starts its cell cycle with a single flagellum. A new flagellum is assembled de novo, while the old flagellum persists throughout the cell cycle. The flagella in an asynchronous L. pyrrhocoris population exhibited a vast length variation of ∼3 to 24 µm, casting doubt on the presence of a length regulation mechanism based on a single balance point between the assembly and disassembly rate in these cells. Through imaging of live L. pyrrhocoris cells, a rapid, partial disassembly of the existing, old flagellum is observed upon, if not prior to, the initial assembly of a new flagellum. Mathematical modeling demonstrated an inverse correlation between the flagellar growth rate and flagellar length and inferred the presence of distinct, cell cycle-dependent disassembly mechanisms with different rates. On the basis of these observations, we proposed a min-max model that could account for the vast flagellar length range observed for asynchronous L. pyrrhocoris. This model may also apply to other flagellated organisms with flagellar length variation.IMPORTANCE Current understanding of flagellum biogenesis during the cell cycle in trypanosomatids is limited to a few pathogenic species, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. The most notable characteristics of trypanosomatid flagella studied so far are the extreme stability and lack of ciliary disassembly/absorption during the cell cycle. This is different from cilia in Chlamydomonas and mammalian cells, which undergo complete absorption prior to cell cycle initiation. In this study, we examined flagellum duplication during the cell cycle of Leptomonas pyrrhocoris With the shortest duplication time documented for all Trypanosomatidae and its amenability to culture on agarose gel with limited mobility, we were able to image these cells through the cell cycle. Rapid, cell cycle-specific flagellum disassembly different from turnover was observed for the first time in trypanosomatids. Given the observed length-dependent growth rate and the presence of different disassembly mechanisms, we proposed a min-max model that can account for the flagellar length variation observed in L. pyrrhocoris.


Assuntos
Ciclo Celular , Flagelos/metabolismo , Trypanosomatina/crescimento & desenvolvimento , Animais , Flagelos/genética , Insetos/parasitologia , Modelos Teóricos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosomatina/genética , Trypanosomatina/metabolismo
8.
Int J Parasitol ; 49(13-14): 1075-1086, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31734337

RESUMO

The extreme biological diversity of Oceanian archipelagos has long stimulated research in ecology and evolution. However, parasitic protists in this geographic area remained neglected and no molecular analyses have been carried out to understand the evolutionary patterns and relationships with their hosts. Papua New Guinea (PNG) is a biodiversity hotspot containing over 5% of the world's biodiversity in less than 0.5% of the total land area. In the current work, we examined insect heteropteran hosts collected in PNG for the presence of trypanosomatid parasites. The diversity of insect flagellates was analysed, to our knowledge for the first time, east of Wallace's Line, one of the most distinct biogeographic boundaries of the world. Out of 907 investigated specimens from 138 species and 23 families of the true bugs collected in eight localities, 135 (15%) were infected by at least one trypanosomatid species. High species diversity of captured hosts correlated with high diversity of detected trypanosomatids. Of 46 trypanosomatid Typing Units documented in PNG, only eight were known from other geographic locations, while 38 TUs (~83%) have not been previously encountered. The widespread trypanosomatid TUs were found in both widely distributed and endemic/sub-endemic insects. Approximately one-third of the endemic trypanosomatid TUs were found in widely distributed hosts, while the remaining species were confined to endemic and sub-endemic insects. The TUs from PNG form clades with conspicuous host-parasite coevolutionary patterns, as well as those with a remarkable lack of this trait. In addition, our analysis revealed new members of the subfamilies Leishmaniinae and Strigomonadinae, potentially representing new genera of trypanosomatids.


Assuntos
Biodiversidade , Insetos/parasitologia , Trypanosomatina/classificação , Trypanosomatina/isolamento & purificação , Animais , Evolução Biológica , Interações Hospedeiro-Parasita , Papua Nova Guiné , Filogenia , Trypanosomatina/genética
9.
PLoS Genet ; 15(11): e1008452, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31710597

RESUMO

Trypanosomatid parasites are causative agents of important human and animal diseases such as sleeping sickness and leishmaniasis. Most trypanosomatids are transmitted to their mammalian hosts by insects, often belonging to Diptera (or true flies). These are called dixenous trypanosomatids since they infect two different hosts, in contrast to those that infect just insects (monoxenous). However, it is still unclear whether dixenous and monoxenous trypanosomatids interact similarly with their insect host, as fly-monoxenous trypanosomatid interaction systems are rarely reported and under-studied-despite being common in nature. Here we present the genome of monoxenous trypanosomatid Herpetomonas muscarum and discuss its transcriptome during in vitro culture and during infection of its natural insect host Drosophila melanogaster. The H. muscarum genome is broadly syntenic with that of human parasite Leishmania major. We also found strong similarities between the H. muscarum transcriptome during fruit fly infection, and those of Leishmania during sand fly infections. Overall this suggests Drosophila-Herpetomonas is a suitable model for less accessible insect-trypanosomatid host-parasite systems such as sand fly-Leishmania.


Assuntos
Interações Hospedeiro-Parasita/genética , Leishmania/genética , Psychodidae/parasitologia , Trypanosomatina/genética , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/parasitologia , Infecções por Euglenozoa/genética , Infecções por Euglenozoa/parasitologia , Infecções por Euglenozoa/transmissão , Humanos , Insetos Vetores/genética , Leishmania/patogenicidade , Leishmaniose/genética , Leishmaniose/parasitologia , Leishmaniose/transmissão , Psychodidae/genética , Trypanosomatina/patogenicidade
10.
Emerg Infect Dis ; 25(11): 2088-2092, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31625841

RESUMO

Through whole-genome sequencing analysis, we identified non-Leishmania parasites isolated from a man with a fatal visceral leishmaniasis-like illness in Brazil. The parasites infected mice and reproduced the patient's clinical manifestations. Molecular epidemiologic studies are needed to ascertain whether a new infectious disease is emerging that can be confused with leishmaniasis.


Assuntos
Infecções por Euglenozoa/epidemiologia , Infecções por Euglenozoa/parasitologia , Trypanosomatina/genética , Idoso , Animais , Brasil/epidemiologia , DNA Espaçador Ribossômico , Genes de Helmintos , Humanos , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Masculino , Camundongos , Filogenia , Trypanosomatina/classificação
11.
Biochim Biophys Acta Mol Cell Res ; 1866(12): 118520, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31369765

RESUMO

Trypanosomatid parasites cause devastating African sleeping sickness, Chagas disease, and Leishmaniasis that affect about 18 million people worldwide. Recently, we showed that the biogenesis of glycosomes could be the "Achilles' heel" of trypanosomatids suitable for the development of new therapies against trypanosomiases. This was shown for inhibitors of the import machinery of matrix proteins, while the distinct machinery for the topogenesis of glycosomal membrane proteins evaded investigation due to the lack of a druggable interface. Here we report on the identification of the highly divergent trypanosomal PEX3, a central component of the transport machinery of peroxisomal membrane proteins and the master regulator of peroxisome biogenesis. The trypanosomatid PEX3 shows very low degree of conservation and its identification was made possible by a combinatory approach identifying of PEX19-interacting proteins and secondary structure homology screening. The trypanosomal PEX3 localizes to glycosomes and directly interacts with the membrane protein import receptor PEX19. RNAi-studies revealed that the PEX3 is essential and that its depletion results in mislocalization of glycosomal proteins to the cytosol and a severe growth defect. Comparison of the parasites and human PEX3-PEX19 interface disclosed differences that might be accessible for drug development. The absolute requirement for biogenesis of glycosomes and its structural distinction from its human counterpart make PEX3 a prime drug target for the development of novel therapies against trypanosomiases. The identification paves the way for future drug development targeting PEX3, and for the analysis of additional partners involved in this crucial step of glycosome biogenesis.


Assuntos
Microcorpos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosomatina/metabolismo , Proteínas de Arabidopsis/metabolismo , Células Cultivadas , Biologia Computacional , Humanos , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Peroxinas/metabolismo , Trypanosomatina/citologia
12.
Int J Parasitol ; 49(8): 605-613, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31163178

RESUMO

The parasite Crithidia mellificae (Kinetoplastea: Trypanosomatidae) infects honeybees, Apis mellifera. No pathogenic effects have been found in individual hosts, despite positive correlations between infections and colony mortalities. The solitary bee Osmia cornuta might constitute a host, but controlled infections are lacking to date. Here, we challenged male and female O. cornuta and honeybee workers in laboratory cages with C. mellificae. No parasite cells were found in any control. Parasite numbers increased 6.6 fold in honeybees between days 6 and 19 p.i. and significantly reduced survival. In O. cornuta, C. mellificae numbers increased 2-3.6 fold within cages and significantly reduced survival of males, but not females. The proportion of infected hosts increased in O. cornuta cages with faeces, but not in honeybee cages without faeces, suggesting faecal - oral transmission. The data show that O. cornuta is a host of C. mellificae and suggest that males are more susceptible. The higher mortality of infected honeybees proposes a mechanism for correlations between C. mellificae infections and colony mortalities.


Assuntos
Criação de Abelhas , Abelhas/parasitologia , Crithidia/fisiologia , Análise de Variância , Animais , Animais Selvagens , Criação de Abelhas/métodos , Abelhas/anatomia & histologia , Tamanho Corporal , Diploide , Feminino , Haploidia , Estimativa de Kaplan-Meier , Masculino , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real , Trypanosomatina/fisiologia
13.
Biol Rev Camb Philos Soc ; 94(5): 1701-1721, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31095885

RESUMO

Parasitic trypanosomatids and phototrophic euglenids are among the most extensively studied euglenozoans. The phototrophic euglenid lineage arose relatively recently through secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga that evolved into the euglenid secondary chloroplast. The parasitic trypanosomatids (i.e. Trypanosoma spp. and Leishmania spp.) and the freshwater phototrophic euglenids (i.e. Euglena gracilis) are the most evolutionary distant lineages in the Euglenozoa phylogenetic tree. The molecular and cell biological traits they share can thus be considered as ancestral traits originating in the common euglenozoan ancestor. These euglenozoan ancestral traits include common mitochondrial presequence motifs, respiratory chain complexes containing various unique subunits, a unique ATP synthase structure, the absence of mitochondria-encoded transfer RNAs (tRNAs), a nucleus with a centrally positioned nucleolus, closed mitosis without dissolution of the nuclear membrane and nucleoli, a nuclear genome containing the unusual 'J' base (ß-D-glucosyl-hydroxymethyluracil), processing of nucleus-encoded precursor messenger RNAs (pre-mRNAs) via spliced-leader RNA (SL-RNA) trans-splicing, post-transcriptional gene silencing by the RNA interference (RNAi) pathway and the absence of transcriptional regulation of nuclear gene expression. Mitochondrial uridine insertion/deletion RNA editing directed by guide RNAs (gRNAs) evolved in the ancestor of the kinetoplastid lineage. The evolutionary origin of other molecular features known to be present only in either kinetoplastids (i.e. polycistronic transcripts, compaction of nuclear genomes) or euglenids (i.e. monocistronic transcripts, huge genomes, many nuclear cis-spliced introns, polyproteins) is unclear.


Assuntos
Evolução Biológica , Euglenozoários/classificação , Biologia Molecular , Trypanosomatina/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Euglênidos/classificação , Euglênidos/genética , Euglenozoários/genética , Genoma/fisiologia , Íntrons/fisiologia , Mitocôndrias/genética , Processos Fototróficos , Filogenia , Interferência de RNA , RNA Ribossômico 28S/genética , Trypanosomatina/classificação , Trypanosomatina/enzimologia
14.
Parasitology ; 146(8): 1036-1046, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064439

RESUMO

In birds, vector-borne parasites invading the bloodstream are important agents of disease, affect fitness and shape population viability, thus being of conservation interest. Here, we molecularly identified protozoan blood parasites in two populations of the threatened Aquatic Warbler Acrocephalus paludicola, a migratory passerine nesting in open marsh. We explored whether prevalence and lineage diversity of the parasites vary by population and whether infection status is explained by landscape metrics of habitat edge and individual traits (body mass, fat score, wing length and sex). Aquatic Warblers were infected by genera Plasmodium, Leucocytozoon and Trypanosoma, with seven, one and four lineages, and 29.9, 0.7 and 12.5% prevalence, respectively. No Haemoproteus infections were detected. Prevalence did not vary between the populations, but lineage diversity was higher in Polesie than in Biebrza for all the lineages pooled and for Plasmodium. Infection by Trypanosoma decreased with patch core area and increased with density of habitat edge. Infection status was not predicted by the individual traits. Our study is the first to show an association between edge-related landscape features and blood parasitism in an open habitat bird. This finding will support informed conservation measures for avian species of the globally shrinking marshland and other treeless habitats.


Assuntos
Ecossistema , Aves Canoras , Trypanosoma/fisiologia , Tripanossomíase/veterinária , Animais , Biodiversidade , Variação Biológica Individual , Plasmodium/isolamento & purificação , Polônia/epidemiologia , Prevalência , Trypanosomatina/isolamento & purificação , Tripanossomíase/epidemiologia , Tripanossomíase/parasitologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-31080782

RESUMO

Two trypanosomatid species, Lotmaria passim and Crithidia mellificae, have been shown to parasitize honey bees to date. L. passim appears to be more prevalent than C. mellificae and specifically infects the honey bee hindgut. Although the genomic DNA has been sequenced, the effects of infection on honey bee health and colony are poorly understood. To identify the genes that are important for infecting honey bees and to understand their functions, we applied the CRISPR/Cas9 system to establish a method to manipulate L. passim genes. By electroporation of plasmid DNA and subsequent selection by drug, we first established an L. passim clone expressing tdTomato or Cas9. We also successfully disrupted the endogenous miltefosine transporter and tyrosine aminotransferase genes by replacement with drug (hygromycin) resistant gene using the CRISPR/Cas9-induced homology-directed repair pathway. The L. passim clone expressing fluorescent marker, as well as the simple method for editing specific genes, could become useful approaches to understand the underlying mechanisms of honey bee-trypanosomatid parasite interactions.


Assuntos
Abelhas/parasitologia , Edição de Genes/métodos , Genética Microbiana/métodos , Trypanosomatina/genética , Animais , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Recombinação Genética
16.
Methods Mol Biol ; 1971: 109-122, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980300

RESUMO

Ribosomes are the machinery responsible for reading mRNAs and translating them into proteins. The ribosome profiling approach is based on high-throughput sequencing of ribosome-protected mRNAs. RNAs not harboring ribosomes are removed by nuclease digestion leaving the so-called ribosome "footprints." The purified "footprint" RNA molecules are processed into DNA libraries and their individual abundance is determined by deep sequencing. Ribosome profiling reveals the portion of transcripts which are actually protein-coding and can be used for differential gene expression analysis addressing rates of protein synthesis, and translational control and efficiency.


Assuntos
Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , RNA Mensageiro/genética , RNA de Protozoário/genética , Ribossomos/genética , Trypanosomatina/genética , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Ribossomos/metabolismo , Trypanosomatina/metabolismo
17.
PLoS One ; 14(4): e0214484, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30943229

RESUMO

Here we described a new trypanosomatid species, Phytomonas lipae, parasitizing the dock bug Coreus marginatus based on axenic culture and in vivo material. Using light and electron microscopy we characterized the development of this flagellate in the intestine, hemolymph and salivary glands of its insect host. The intestinal promastigotes of Phytomonas lipae do not divide and occur only in the anterior part of the midgut. From there they pass into hemolymph, increasing in size, and then to salivary glands, where they actively proliferate without attachment to the host's epithelium and form infective endomastigotes. We conducted molecular phylogenetic analyses based on 18s rRNA, gGAPDH and HSP83 gene sequences, of which the third marker performed the best in terms of resolving phylogenetic relationships within the genus Phytomonas. Our inference demonstrated rather early origin of the lineage comprising the new species, right after that of P. oxycareni, which represents the earliest known branch within the Phytomonas clade. This allowed us to compare the development of P. lipae and three other Phytomonas spp. in their insect hosts and reconstruct the vectorial part of the life cycle of their common ancestor.


Assuntos
Heterópteros/parasitologia , Estágios do Ciclo de Vida , Glândulas Salivares/parasitologia , Trypanosomatina/genética , Animais , Proteínas de Choque Térmico/genética , Intestinos/parasitologia , Kinetoplastida , Funções Verossimilhança , Filogenia , Proteínas de Protozoários/genética , RNA Ribossômico 18S/genética , Trypanosomatina/classificação , Trypanosomatina/fisiologia
18.
PLoS Genet ; 15(3): e1007931, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30822306

RESUMO

Dipteran insects transmit serious diseases to humans, often in the form of trypanosomatid parasites. To accelerate research in more difficult contexts of dipteran-parasite relationships, we studied the interaction of the model dipteran Drosophila melanogaster and its natural trypanosomatid Herpetomonas muscarum. Parasite infection reduced fecundity but not lifespan in NF-κB/Relish-deficient flies. Gene expression analysis implicated the two NF-κB pathways Toll and Imd as well as STAT signalling. Tissue specific knock-down of key components of these pathways in enterocytes (ECs) and intestinal stem cells (ISCs) influenced initial numbers, infection dynamics and time of clearance. Herpetomonas triggered STAT activation and proliferation of ISCs. Loss of Relish suppressed ISCs, resulting in increased parasite numbers and delayed clearance. Conversely, overexpression of Relish increased ISCs and reduced uptake. Finally, loss of Toll signalling decreased EC numbers and enabled parasite persistence. This network of signalling may represent a general mechanism with which dipteran respond to trypanosomatids.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Interações Hospedeiro-Parasita/genética , Fatores de Transcrição/genética , Trypanosomatina/genética , Animais , Proliferação de Células/genética , Drosophila melanogaster/parasitologia , Enterócitos/metabolismo , Enterócitos/parasitologia , Fertilidade/genética , Regulação da Expressão Gênica/genética , Humanos , Intestinos/parasitologia , Fatores de Transcrição STAT/genética , Transdução de Sinais/genética , Células-Tronco/metabolismo , Receptores Toll-Like/genética , Fator de Transcrição RelA/genética , Trypanosomatina/patogenicidade
19.
Parasite ; 26: 17, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30901308

RESUMO

The measurement of respiratory chain enzyme activities is an integral part of basic research as well as for specialized examinations in clinical biochemistry. Most of the enzymes use ubiquinone as one of their substrates. For current in vitro measurements, several hydrophilic analogues of native ubiquinone are used depending on the enzyme and the workplace. We tested five readily available commercial analogues and we showed that Coenzyme Q2 is the most suitable for the measurement of all tested enzyme activities. Use of a single substrate in all laboratories for several respiratory chain enzymes will improve our ability to compare data, in addition to simplifying the stock of chemicals required for this type of research.


Assuntos
Trypanosomatina/enzimologia , Ubiquinona/análogos & derivados , Transporte de Elétrons , Ubiquinona/metabolismo
20.
Infect Genet Evol ; 70: 90-100, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30738194

RESUMO

The mitochondrial DNA (mtDNA) is a potentially valuable phylogenetic marker given its presence across all eukaryotic taxa and its relative conservation in structure and sequence. In trypanosomatids, a homologue of the mtDNA referred to as the maxicircle DNA, is located within a specialised structure in the single mitochondrion of the trypanosomatids called the kinetoplast; a high molecular weight network of DNA composed of thousands of catenated minicircles and a smaller number of larger maxicircles. Unique to the kinetoplastid protists, the maxicircle component of this complex network could represent a desirable target for taxonomic inquiry that may also facilitate exploration of the evolutionary history of this important group of parasites. The aim of this study was to investigate the phylogenetic value of the trypanosomatid maxicircle for these applications. Maxicircle sequences were obtained either by assembling raw sequence data publicly accessible in online databases (i.e., NCBI), or by amplification of novel maxicircle sequences from trypanosomatid DNA using long-range (LR) PCR with subsequent Illumina sequencing. This procedure facilitated the generation of nearly complete maxicircle sequences (i.e., excluding the divergent region) for numerous dixenous and monoxenous trypanosomatid species. Annotation of each maxicircle sequence confirmed that their structure was conserved across all taxa examined. Phylogenetic analyses confirmed that Z. australiensis showed a greater genetic relatedness with the dixenous trypanosomatids of the genera Leishmania and Endotrypanum, as opposed to members of the monoxenous genera Crithidia and Leptomonas. Additionally, molecular clock analysis supported that the dixenous Leishmaniinae appeared approximately 75 million years ago during the breakup of Gondwana. In line with previous studies, our results support the Supercontinents hypothesis regarding the origin of dixenous Leishmaniinae. Ultimately, we demonstrate that the maxicircle represents an excellent phylogenetic marker for studying the evolutionary history of trypanosomatids, resulting in trees with very high bootstrap support values.


Assuntos
DNA de Cinetoplasto/genética , Trypanosomatina/genética , Animais , Evolução Biológica , Crithidia/genética , Crithidia/ultraestrutura , Marcadores Genéticos , Leishmania/genética , Leishmania/ultraestrutura , Filogeografia , Trypanosomatina/ultraestrutura
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