Imidacloprid increases the prevalence of the intestinal parasite Lotmaria passim in honey bee workers.

A challenge in bee protection is to assess the risks of pesticide-pathogen interactions. Lotmaria passim, a ubiquitous unicellular parasite in honey bees, is considered harmful under specific conditions. Imidacloprid causes unpredictable side effects. Research indicates that both L. passim and imidacloprid may affect the physiology, behavior, immunity, microbiome and lifespan of honey bees. We designed cage experiments to test whether the infection of L. passim is affected by a sublethal dose of imidacloprid. Workers collected at the time of emergence were exposed to L. passim and 2.5 µg/L imidacloprid in the coexposure treatment group. First, samples of bees were taken from cages since they were 5 days old and 3 days postinfection, i.e., after finishing an artificial 24 h L. passim infection. Additional bees were collected every two additional days. In addition, bees frozen at the time of emergence and collected from the unexposed group were analyzed. Abdomens were analyzed using qPCR to determine parasite load, while corresponding selected heads were subjected to a label-free proteomic analysis. Our results show that bees are free of L. passim at the time of emergence. Furthermore, imidacloprid considerably increased the prevalence as well as parasite loads in individual bees. This means that imidacloprid facilitates infection, enabling faster parasite spread in a colony and potentially to surrounding colonies. The proteomic analysis of bee heads showed that imidacloprid neutralized the increased transferrin 1 expression by L. passim. Importantly, this promising marker has been previously observed to be upregulated by infections, including gut parasites. This study contributes to understanding the side effects of imidacloprid and demonstrates that a single xenobiotic/pesticide compound can interact with the gut parasite. Our methodology can be used to assess the effects of different compounds on L. passim.

Diversity of RNA viruses in the cosmopolitan monoxenous trypanosomatid Leptomonas pyrrhocoris.

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.

Shining the spotlight on the neglected: new high-quality genome assemblies as a gateway to understanding the evolution of Trypanosomatidae.

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.

Frequent Parasitism of Apis mellifera by Trypanosomatids in Geographically Isolated Areas with Restricted Beekeeping Movements.

Trypanosomatids form a group of high prevalence protozoa that parasitise honey bees, with Lotmaria passim as the predominant species worldwide. However, the knowledge about the ecology of trypanosomatids in isolated areas is limited. The Portuguese archipelagos of Madeira and Azores provide an interesting setting to investigate these parasites because of their geographic isolation, and because they harbour honey bee populations devoid of two major enemies: Varroa destructor and Nosema ceranae. Hence, a total of 661 honey bee colonies from Madeira and the Azores were analysed using different molecular techniques, through which we found a high prevalence of trypanosomatids despite the isolation of these islands. L. passim was the predominant species and, in most colonies, was the only one found, even on islands free of V. destructor and/or N. ceranae with severe restrictions on colony movements to prevent the spread of them. However, islands with V. destructor had a significantly higher prevalence of L. passim and, conversely, islands with N. ceranae did not shown any significant correlation with the trypanosomatid. Crithidia bombi was detected in Madeira and on three islands of the Azores, almost always coincident with L. passim. By contrast, Crithidia mellificae was not detected in any sample. A high-throughput sequencing analysis distinguished two main haplotypes of L. passim, which accounted for 98% of the total sequence reads. This work suggests that L. passim and C. bombi are parasites that have been associated with honey bees predating the spread of V. destructor and N. ceranae.

A new class of antiparasitic drugs.

Cyanotriazole compounds "poison" topoisomerase II of pathogenic trypanosomatids.

A neo-functionalized homolog of host transmembrane protein controls localization of bacterial endosymbionts in the trypanosomatid Novymonas esmeraldas.

The stability of endosymbiotic associations between eukaryotes and bacteria depends on a reliable mechanism ensuring vertical inheritance of the latter. Here, we demonstrate that a host-encoded protein, located at the interface between the endoplasmic reticulum of the trypanosomatid Novymonas esmeraldas and its endosymbiotic bacterium Ca. Pandoraea novymonadis, regulates such a process. This protein, named TMP18e, is a product of duplication and neo-functionalization of the ubiquitous transmembrane protein 18 (TMEM18). Its expression level is increased at the proliferative stage of the host life cycle correlating with the confinement of bacteria to the nuclear vicinity. This is important for the proper segregation of bacteria into the daughter host cells as evidenced from the TMP18e ablation, which disrupts the nucleus-endosymbiont association and leads to greater variability of bacterial cell numbers, including an elevated proportion of aposymbiotic cells. Thus, we conclude that TMP18e is necessary for the reliable vertical inheritance of endosymbionts.

Minimal sharing of nosematid and trypanosomatid parasites between honey bees and other bees, but extensive sharing of Crithidia between bumble and mason bees.

We document gut parasites in co-occurring Apis, Bombus, and Osmia spp. in the Northern Virginia region, USA. Trypanosomatidea occurred in sixty percent of specimens and 13% carried Nosematidae. We found strong host partitioning: Lotmaria passim and Vairimorpha (Nosema) ceranae predominated in Apis, and Crithidia bombi and V. bombi in Bombus. We did not detect pathogen spread from Apis to Bombus but did detect sharing of C. bombi between Bombus and Osmia, higher parasite levels in Apis at sites with apiaries, and clustering of Vairimopha infection. Given the presence of C. bombi in Osmia, we suggest disease sharing across taxa be monitored.

Structural design, synthesis, and anti-Trypanosomatidae profile of new Pyridyl-thiazolidinones.

The present work reports the synthesis of a novel series of pyridine-thiazolidinones with anti-Trypanosoma cruzi and leishmanicidal activities (compounds 10-27), derived from 2 or 4-pyridine thiosemicarbazones (1-9). The in vitro assays were performed with Trypanosoma cruzi trypomastigotes and amastigotes, as well as with Leishmania amazonensis promastigotes and amastigotes. The cytotoxicity profile was evaluated using the cell line RAW 264.7. From the 18 pyridine-thiazolidinones, 5 were able to inhibit trypomastigotes. Overall, all compounds inhibited amastigotes, highlighting compounds 15 (0.60 µM), 18 (0.64 µM), 17 (0.81 µM), and 27 (0.89 µM). Compounds 15 and 18 were able to induce parasite cell death through necrosis induction. Analysis by scanning electron microscopy showed that T. cruzi trypomastigotes treated with compounds 15 and 18 induced morphological changes such as shortening, retraction and curvature of the parasite body and leakage of internal content. Regarding the antiparasitic evaluation against Leishmania amazonensis, only compound 27 had a higher selectivity compared to Miltefosine against the amastigote form (IC50 = 5.70 µM). Our results showed that compound 27 presented an antiparasitic activity for both Trypanosoma cruzi and Leishmania amazonensis. After in silico evaluation, it was suggested that the new pyridine-thiazolidinones had an appropriate drug-likeness profile. Our results pointed out a new chemical frame with an anti-Trypanosomatidae profile. The pyridine-thiazolidinones presented here for the first time could be used as a starting point for the development of new antiparasitic agents.

Anti-Trypanosomatidae Activity of Essential Oils and Their Main Components from Selected Medicinal Plants.

Kinetoplastida is a group of flagellated protozoa characterized by the presence of a kinetoplast, a structure which is part of a large mitochondria and contains DNA. Parasites of this group include genera such as Leishmania, that cause disease in humans and animals, and Phytomonas, that are capable of infecting plants. Due to the lack of treatments, the low efficacy, or the high toxicity of the employed therapeutic agents there is a need to seek potential alternative treatments. In the present work, the antiparasitic activity on Leishmania infantum and Phytomonas davidi of 23 essential oils (EOs) from plants of the Lamiaceae and Asteraceae families, extracted by hydrodistillation (HD) at laboratory scale and steam distillation (SD) in a pilot plant, were evaluated. The chemical compositions of the EOs were determined by gas chromatography-mass spectrometry. Additionally, the cytotoxic activity on mammalian cells of the major components from the most active EOs was evaluated, and their anti-Phytomonas and anti-Leishmania effects analyzed. L. infantum was more sensitive to the EOs than P. davidi. The EOs with the best anti-kinetoplastid activity were S. montana, T. vulgaris, M. suaveolens, and L. luisieri. Steam distillation increased the linalyl acetate, ß-caryophyllene, and trans-α-necrodyl acetate contents of the EOs, and decreased the amount of borneol and 1,8 cineol. The major active components of the EOs were tested, with thymol being the strongest anti-Phytomonas compound followed by carvacrol. Our study identified potential treatments against kinetoplastids.

Development of a TaqMan qPCR assay for trypanosomatid multi-species detection and quantification in insects.

BACKGROUND: Trypanosomatid parasites are widely distributed in nature and can have a monoxenous or dixenous life-cycle. These parasites thrive in a wide number of insect orders, some of which have an important economic and environmental value, such as bees. The objective of this study was to develop a robust and sensitive real-time quantitative PCR (qPCR) assay for detecting trypanosomatid parasites in any type of parasitized insect sample. METHODS: A TaqMan qPCR assay based on a trypanosomatid-conserved region of the α-tubulin gene was standardized and evaluated. The limits of detection, sensitivity and versatility of the α-tubulin TaqMan assay were tested and validated using field samples of honeybee workers, wild bees, bumblebees and grasshoppers, as well as in the human infective trypanosomatid Leishmania major. RESULTS: The assay showed a detection limit of 1 parasite equivalent/µl and successfully detected trypanosomatids in 10 different hosts belonging to the insect orders Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from four apiaries (n = 224 worker honeybees) located in the Alpujarra region (Granada, Spain). Trypanosomatids were detected in 2.7% of the honeybees, with an intra-colony prevalence of 0% to 13%. Parasite loads in the four different classes of insects ranged from 40.6 up to 1.1 × 108 cell equivalents per host. CONCLUSIONS: These results show that the α-tubulin TaqMan qPCR assay described here is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatids in a wide range of environmental settings.

Host-symbiont interactions in Angomonas deanei include the evolution of a host-derived dynamin ring around the endosymbiont division site.

The trypanosomatid Angomonas deanei is a model to study endosymbiosis. Each cell contains a single ß-proteobacterial endosymbiont that divides at a defined point in the host cell cycle and contributes essential metabolites to the host metabolism. Additionally, one endosymbiont gene, encoding an ornithine cyclodeaminase (OCD), was transferred by endosymbiotic gene transfer (EGT) to the nucleus. However, the molecular mechanisms mediating the intricate host/symbiont interactions are largely unexplored. Here, we used protein mass spectrometry to identify nucleus-encoded proteins that co-purify with the endosymbiont. Expression of fluorescent fusion constructs of these proteins in A. deanei confirmed seven host proteins to be recruited to specific sites within the endosymbiont. These endosymbiont-targeted proteins (ETPs) include two proteins annotated as dynamin-like protein and peptidoglycan hydrolase that form a ring-shaped structure around the endosymbiont division site that remarkably resembles organellar division machineries. The EGT-derived OCD was not among the ETPs, but instead localizes to the glycosome, likely enabling proline production in the glycosome. We hypothesize that recalibration of the metabolic capacity of the glycosomes that are closely associated with the endosymbiont helps to supply the endosymbiont with metabolites it is auxotrophic for and thus supports the integration of host and endosymbiont metabolic networks. Hence, scrutiny of endosymbiosis-induced protein re-localization patterns in A. deanei yielded profound insights into how an endosymbiotic relationship can stabilize and deepen over time far beyond the level of metabolite exchange.

Does Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida: Trypanosomatidae) modify the antennal phenotype of Triatoma dimidiata (Latreille, 1811) (Hemiptera: Triatominae)?

BACKGROUND: Triatoma dimidiata is a vector of the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease. Phenotypic plasticity allows an organism to adjust its phenotype in response to stimuli or environmental conditions. Understanding the effect of T. cruzi on the phenotypic plasticity of its vectors, known as triatomines, has attracted great interest because of the implications of the parasite-triatomine interactions in the eco-epidemiology and transmission of the etiologic agent of Chagas disease. We investigated if the infection of the vector with T. cruzi may be associated with a change in the antennal phenotype of sylvatic, domestic, and laboratory-reared populations of T. dimidiata. METHODS: The abundance of each type of sensillum (bristles, basiconic, thick- and thin-walled trichoid) on the antennae of T. cruzi-infected and non-infected T. dimidiata reared in the laboratory or collected in sylvatic and domestic ecotopes were measured under light microscopy and compared using Kruskal-Wallis non-parametric tests and permutational multivariate analysis of variance. RESULTS: We found significant differences between sensilla patterns of infected and non-infected insects within sylvatic and domestic populations. Conversely, we found no significant differences between sensilla patterns of infected and non-infected insects within the laboratory-reared population. Besides, for sylvatic and domestic populations, sexual dimorphism tended to be increased in infected insects. CONCLUSION: The differences observed in infected insects could be linked to higher efficiency in the perception of odor molecules related to the search for distant mates and hosts and the flight dispersal in search of new habitats. In addition, these insects could have a positive effect on population dynamics and the transmission of T. cruzi.

Interrogating the transmission dynamics of Trypanosoma cruzi (Trypanosomatida, Trypanosomatidae) by Triatoma venosa (Hemiptera: Reduviidae) after the elimination of vector transmission by Rhodnius prolixus in Boyacá eastern Colombia.

Chagas disease (CD) is a parasitic zoonosis (Trypanosoma cruzi) that is endemic in Colombia. Vector control of Rhodnius prolixus, the main domestic T. cruzi vector, has been achieved in a large part of the area with historically vector transmission of CD. It is necessary to understand the ecological behavior characteristics of local native vectors to ensure sustainability of the vector control programs. To evaluate the long-term success of a recent vector control campaign in the Boyacá department (Colombia), we used a combined strategy of entomological surveillance with co-existing canine surveillance from ten rural villages within six municipalities of the Tenza valley region (Boyacá, Colombia): Chinavita, Garagoa, Guateque, Somondoco, Sutatenza and Tenza, with historical reports of R. prolixus and secondary vectors. Collected triatomines and canine whole blood were analyzed for T. cruzi infection and genotyping. Triatomine bugs specimens were evaluated for blood meal source. Canine serology was performed using two distinct antibody assays. In total, 101 Triatoma venosa were collected by active search in domestic and peridomestic habitats. A natural infection prevalence of 13.9% (14/101) and four feeding sources were identified: human, dog, rat, and hen. A frequency infection of 46.5% (40/87) was observed from two independent serological tests and T. cruzi DNA was detected in 14 dogs (16.4%). Only TcI_sylvatic DTU was detected. The results suggest that T. venosa present eco-epidemiological characteristics to maintain the transmission of T. cruzi in Tenza valley. This species has reinfested the intervened households and it has an active role in domestic and peridomestic transmission of T. cruzi due to their infection rates and feeding behavior. Therefore, this species should be considered as epidemiologically relevant for vector control strategies. Moreover, there is a need for human serological studies to have a close up of risk they are exposed to.

Detection of Leptomonas seymouri narna-like virus in serum samples of visceral leishmaniasis patients and its possible role in disease pathogenesis.

Kala-azar/Visceral Leishmaniasis (VL) caused by Leishmania donovani (LD) is often associated with Leptomonas seymouri (LS) co-infection in India. Leptomonas seymouri narna-like virus 1 (Lepsey NLV1) has been reported in multi-passaged laboratory isolates of VL samples which showed LD-LS co-infection. A pertinent question was whether this virus of LS is detectable in direct clinical samples. DNA from the serum of twenty-eight LD diagnosed patients was subjected to LD-specific and LS-specific PCR to reconfirm the presence of LD parasites and to detect LD-LS co-infections. RNA extracted from same samples was subjected to RT-PCR, qRT-PCR and sequencing using virus-specific primers to detect/identify and quantify the virus. The presence of the virus was confirmed in thirteen of eighteen (72%) recently collected VL and PKDL samples. Cytokine profiling showed significantly elevated IL-18 in only LD infected patients compared to the virus-positive LD and control samples. IL-18 is crucial for Th1 and macrophage activation which eventually clears the parasite. The Lepsey NLV1 interaction with the immune system results in reduced IL-18 which favors LD survival and increased parasitic burden. The study emphasizes the need to revisit LD pathogenesis in the light of the association and persistence of a protozoan virus in kala-azar and PKDL patients.

Isothermal Nucleic Acid Amplification to Detect Infection Caused by Parasites of the Trypanosomatidae Family: A Literature Review and Opinion on the Laboratory to Field Applicability.

Isothermal amplification of nucleic acids has the potential to be applied in resource-limited areas for the detection of infectious agents, as it does not require complex nucleic purification steps or specific and expensive equipment and reagents to perform the reaction and read the result. Since human and animal infections by pathogens of the Tryponasomatidae family occur mainly in resource-limited areas with scant health infrastructures and personnel, detecting infections by these methodologies would hold great promise. Here, we conduct a narrative review of the literature on the application of isothermal nucleic acid amplification for Trypanosoma and Leishmania infections, which are a scourge for human health and food security. We highlight gaps and propose ways to improve them to translate these powerful technologies into real-world field applications for neglected human and animal diseases caused by Trypanosomatidae.

Trypanosomatid Richness Among Rats, Opossums, and Dogs in the Caatinga Biome, Northeast Brazil, a Former Endemic Area of Chagas Disease.

Parasites are important components of the immense n-dimensional trophic network that connects all living beings because they, among others, forge biodiversity and deeply influence ecological evolution and host behavior. In this sense, the influence of Trypanosomatidae remains unknown. The aim of this study was to determine trypanosomatid infection and richness in rats, opossums, and dogs in the semiarid Caatinga biome. We submitted DNA samples from trypanosomatids obtained through axenic cultures of the blood of these mammals to mini exon multiplex-PCR, Sanger, and next-generation sequencing targeting the 18S rDNA gene. Phylogenetic analyses were performed to identify genetic diversity in the Trypanosomatidae family. Shannon, Simpson, equability, and beta-diversity indices were calculated per location and per mammalian host. Dogs were surveyed for trypanosomatid infection through hemocultures and serological assays. The examined mammal species of this area of the Caatinga biome exhibited an enormous trypanosomatid species/genotypes richness. Ten denoised Operational Taxonomic Units (ZOTUs), including three species (Trypanosoma cruzi, Trypanosoma rangeli and Crithidia mellificae) and one Trypanosoma sp. five genotypes/lineages (T. cruzi DTU TcI, TcII, and TcIV; T. rangeli A and B) and four DTU TcI haplotypes (ZOTU1, ZOTU2, ZOTU5, and ZOTU10 merged), as well as 13 Amplicon Sequence Variants (ASVs), including five species (T. cruzi, T. rangeli, C. mellificae, Trypanosoma dionisii, and Trypanosoma lainsoni), five genotypes/lineages (same as the ZOTUs) and six DTU TcI haplotypes (ASV, ASV1, ASV2, ASV3, ASV5 and ASV13), were identified in single and mixed infections. We observed that trypanosomatids present a broad host spectrum given that species related to a single host are found in other mammals from different taxa. Concomitant infections between trypanosomatids and new host-parasite relationships have been reported, and this immense diversity in mammals raised questions, such as how this can influence the course of the infection in these animals and its transmissibility. Dogs demonstrated a high infection rate by T. cruzi as observed by positive serological results (92% in 2005 and 76% in 2007). The absence of positive parasitological tests confirmed their poor infectivity potential but their importance as sentinel hosts of T. cruzi transmission.

Infection susceptibility and vector competence of Rhodnius robustus Larrousse, 1927 and R. pictipes Stal, 1872 (Hemiptera, Reduviidae, Triatominae) for strains of Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae) I, II and IV.

BACKGROUND: Rhodnius robustus and Rhodnius pictipes are vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease (CD), that are found in the Brazilian Amazon region. Susceptibility to infection and vector competence depend on the parasite-vector relationship. Our objective was to evaluate the interaction between T. cruzi and these two triatomine vectors in pure and mixed experimental infections of T. cruzi strains from the same or different geographic regions. METHODS: Fifth-instar nymphs of R. robustus and R. pictipes were fed on mice infected with four T. cruzi strains, namely genotypes TcIAM, TcIMG, TcIIPR, and TcIVAM, respectively, from the Brazilian states of Amazonas, Minas Gerais and Paraná. Over a period of 120 days, excreta were examined every 20 days to assess vector competence, and intestinal contents (IC) were examined every 30 days to determine susceptibility to infection. RESULTS: The highest positive rate in the fresh examination (%+FE, 30.0%), the highest number of parasitic forms (PF, n = 1969) and the highest metacyclogenesis rate (%MC, 53.8%) in the excreta were recorded for R. robustus/TcIVAM. Examination of the IC of R. pictipes revealed a higher number of PF in infections with TcIAM (22,680 PF) and TcIIPR (19,845 PF) alone or in association (17,145 PF), as well as a %+FE of 75.0% with TcII, in comparison with the other genotypes. The highest %MC (100%) was recorded for the mixed infections of TcIAM with TcIIPR or TcIVAM in the IC of R. pictipes. CONCLUSIONS: Overall, both species were found to be susceptible to the T. cruzi strains studied. Rhodnius robustus showed vector competence for genotypes TcIVAM and TcIAM+TcIVAM and R. pictipes for TcIAM+TcIVAM and TcIAM+TcIIPR; there was elimination of infective forms as early as at 20 days. Our results suggest that both the genetics of the parasite and its geographic origin influence the susceptibility to infection and vector competence, alone or in association.

Effect of the endoplasmic reticulum stressor tunicamycin in Angomonas deanei heat-shock protein expression and on the association with the endosymbiotic bacterium.

The endoplasmic reticulum (ER) presents unique properties to establishing bacterium symbiosis in eukaryotic cells since it synthesizes and glycosylates essential molecules like proteins and lipids. Tunicamycin (TM) is an antibiotic that inhibits the first step in the N-linked glycosylation in eukaryotes and has been used as an ER stress inducer to activate the Unfolded Protein Response (UPR). Mutualistic symbiosis in trypanosomatids is characterized by structural adaptations and intense metabolic exchanges, thus we investigated the effects of TM in the association between Angomonas deanei and its symbiotic bacterium, through ultrastructural and proteomic approaches. Cells treated with the inhibitor showed a decrease in proliferation, enlargement of the ER and Golgi cisternae and an increased distance between the symbiont and the ER. TM proved to be an important tool to better understand ER stress in trypanosomatids, since changes in protein composition were observed in the host protozoan, especially the expression of the Hsp90 chaperone. Furthermore, data obtained indicates the importance of the ER for the adaptation and maintenance of symbiotic associations between prokaryotes and eukaryotes, considering that this organelle has recognized importance in the biogenesis and division of cell structures.

Intracellular localization of MyosinXXI discriminates Leishmania spp and Leptomonasseymouri.

The patients with the most dreaded Leishmania donovani infections are now regularly been detected with co-infecting monoxenous trypanosomatid, Leptomonas seymouri, of which pathological consequence is obscure. Due to high degree of morphological similarity, its presence remains unmarked in the culture which leads to anomalous research outcomes. The available methods to detect Leptomonas in cultures are cumbersome and are not quantitative. We report here that MyosinXXI serves as a distinguishing biomarker that can be used to mark the presence of L. seymouri in Leishmania cultures. The method uses Leishmania MyosinXXI antibodies employed in immunofluorescence microscopy that shows a specialized localization pattern in Leishmania but not in Leptomonas (Patent application No. IN201711014439). This method is not only qualitative, but can also quantify the L. seymouri load in the cultured field isolates and serves as a remarkable tool to ascertain laboratory strains of Leishmania.

Molecular detection of Trypanosoma (Trypanosomatidae) in bats from Thailand, with their phylogenetic relationships.

The vast majority of trypanosome species is vector-borne parasites, with some of them being medically and veterinary important (such as Trypanosoma cruzi and Trypanosoma brucei) and capable of causing serious illness in vertebrate hosts. The discovery of trypanosomes in bats emphasizes the importance of bats as an important reservoir. Interestingly, there is a hypothesis that bats are ancestral hosts of T. cruzi. Trypanosome diversity has never been investigated in bats in Thailand, despite being in a biodiversity hot spot. To gain a better understanding of the diversity and evolutionary relationship of trypanosomes, polymerase chain reaction-based surveys were carried out from 2018 to 2020 in 17 sites. A total of 576 bats were captured, representing 23 species. A total of 38 (6.6%) positive samples was detected in ten bat species. Trypanosoma dionisii and Trypanosoma noyesi were identified from Myotis siligorensis and Megaderma spasma, respectively. The remaining 18S rRNA sequences of trypanosomes were related to other trypanosomes previously reported elsewhere. The sequences in the current study showed nucleotide identity as low as 90.74% compared to those of trypanosomes in the GenBank database, indicating the possibility of new species. All bat trypanosomes identified in the current study fall within the T. cruzi clade. The current study adds to evidence linking T. noyesi to a bat trypanosome and further supports the bat host origin of the T. cruzi clade. To the best of authors' knowledge, this is the first study on bat trypanosomes in Thailand and their phylogenetic relationships with global isolates.