Using average nucleotide identity (ANI) to evaluate microsporidia species boundaries based on their genetic relatedness.

Microsporidia are obligatory intracellular parasites related to fungi and since their discovery their classification and origin has been controversial due to their unique morphology. Early taxonomic studies of microsporidia were based on ultrastructural spore features, characteristics of their life cycle and transmission modes. However, taxonomy and phylogeny based solely on these characteristics can be misleading. SSU rRNA is a traditional marker used in taxonomical classifications, but the power of SSU rRNA to resolve phylogenetic relationships between microsporidia is considered weak at the species level, as it may not show enough variation to distinguish closely related species. Overall genome relatedness indices (OGRI), such as average nucleotide identity (ANI), allows fast and easy-to-implement comparative measurements between genomes to assess species boundaries in prokaryotes, with a 95% cutoff value for grouping genomes of the same species. Due to the increasing availability of complete genomes, metrics of genome relatedness have been applied for eukaryotic microbes taxonomy such as microsporidia. However, the distribution of ANI values and cutoff values for species delimitation have not yet been fully tested in microsporidia. In this study we examined the distribution of ANI values for 65 publicly available microsporidian genomes and tested whether the 95% cutoff value is a good estimation for circumscribing species based on their genetic relatedness.

A new microsporidian parasite, Ordospora pajunii sp. nov (Ordosporidae), of Daphnia longispina highlights the value of genomic data for delineating species boundaries.

Speciation is a complex and continuous process that makes the delineation of species boundaries a challenging task in particular in species with little morphological differentiation, such as parasites. In this case, the use of genomic data is often necessary, such as for the intracellular Microsporidian parasites. Here, we characterize the genome of a gut parasite of the cladoceran Daphnia longispina (isolate FI-F-10), which we propose as a new species within the genus Ordospora: Ordospora pajunii sp. nov (Ordosporidae). FI-F-10 closest relative, Ordospora colligata is only found in D. magna. Both microsporidian species share several morphological features. Although it is not possible to estimate divergence times for Microsporidia due to the lack of fossil records and accelerated evolutionary rates, we base our proposal on the phylogenomic and genomic distances between both microsporidian lineages. The phylogenomic reconstruction shows that FI-F-10 forms an early diverging branch basal to the cluster that contains all known O. colligata strains. Whole-genome comparisons show that FI-F-10 presents a greater divergence at the sequence level than observed among O. colligata strains, and its genomic average nucleotide identity (ANI) values against O. colligata are beyond the intraspecific range previously established for yeast and prokaryotes. Our data confirm that the ANI metrics are useful for fine genetic divergence calibration across Microsporidia taxa. In combination with phylogenetic and ecological data, genome-based metrics provide a powerful approach to delimitate species boundaries.

Microsporidia with Vertical Transmission Were Likely Shaped by Nonadaptive Processes.

Microsporidia have the leanest genomes among eukaryotes, and their physiological and genomic simplicity has been attributed to their intracellular, obligate parasitic life-style. However, not all microsporidia genomes are small or lean, with the largest dwarfing the smallest ones by at least an order of magnitude. To better understand the evolutionary mechanisms behind this genomic diversification, we explore here two clades of microsporidia with distinct life histories, Ordospora and Hamiltosporidium, parasitizing the same host species, Daphnia magna. Based on seven newly assembled genomes, we show that mixed-mode transmission (the combination of horizontal and vertical transmission), which occurs in Hamiltosporidium, is found to be associated with larger and AT-biased genomes, more genes, and longer intergenic regions, as compared with the exclusively horizontally transmitted Ordospora. Furthermore, the Hamiltosporidium genome assemblies contain a variety of repetitive elements and long segmental duplications. We show that there is an excess of nonsynonymous substitutions in the microsporidia with mixed-mode transmission, which cannot be solely attributed to the lack of recombination, suggesting that bursts of genome size in these microsporidia result primarily from genetic drift. Overall, these findings suggest that the switch from a horizontal-only to a mixed mode of transmission likely produces population bottlenecks in Hamiltosporidium species, therefore reducing the effectiveness of natural selection, and allowing their genomic features to be largely shaped by nonadaptive processes.

Triatomine bugs, their microbiota and Trypanosoma cruzi: asymmetric responses of bacteria to an infected blood meal.

BACKGROUND: Triatomine bugs (Hemiptera: Reduviidae) are vectors of the flagellate Trypanosoma cruzi, the causative agent of Chagas disease. The study of triatomine gut microbiota has gained relevance in the last years due to its possible role in vector competence and prospective use in control strategies. The objective of this study is to examine changes in the gut microbiota composition of triatomines in response to a T. cruzi-infected blood meal and identifying key factors determining those changes. RESULTS: We sampled colony-reared individuals from six triatomine vectors (Panstrongylus megistus, Rhodnius prolixus, Triatoma brasiliensis, T. infestans, T. juazeirensis and T. sherlocki) comparing experimentally T. cruzi strain 0354-challenged and non-challenged insects. The microbiota of gut and gonad tissues was characterized using high throughput sequencing of region V3-V4 of bacterial 16S rRNA gene. The triatomine microbiota had a low intra-individual diversity, and a high inter-individual variation within the same host species. Arsenophonous appeared as the dominant triatomine bacterial symbiont in our study (59% of the total 16S coverage), but there were significant differences in the distribution of bacterial genera among vectors. In Rhodnius prolixus the dominant symbiont was Pectobacterium. CONCLUSIONS: Trypanosoma cruzi-challenge significantly affects microbiota composition, with challenged vectors harbouring a significantly more diverse bacterial community, both in the gut and the gonads. Our results show that blood-feeding with T. cruzi epimastigotes strongly affects microbiota composition in a species-specific manner. We suggest that triatomine-adapted enterobacteria such as Arsenophonus could be used as stable vectors for genetic transformation of triatomine bugs and control of Chagas disease.

Mitochondrial and nuclear sequence polymorphisms reveal geographic structuring in Amazonian populations of Echinococcus vogeli (Cestoda: Taeniidae).

To date, nothing is known about the genetic diversity of the Echinococcus neotropical species, Echinococcus vogeli and Echinococcus oligarthrus. Here we used mitochondrial and nuclear DNA sequence polymorphisms to uncover the genetic structure, transmission and history of E. vogeli in the Brazilian Amazon, based on a sample of 38 isolates obtained from human and wild animal hosts. We confirm that the parasite is partially synanthropic and show that its populations are diverse. Furthermore, significant geographical structuring is found, with western and eastern populations being genetically divergent.

Echinococcus ortleppi (G5) and Echinococcus granulosus sensu stricto (G1) loads in cattle from Southern Brazil.

Echinococcus granulosus sensu stricto (G1) and Echinococcus ortleppi (G5) are haplotypes of the parasite formerly known as Echinococcus granulosus sensu lato, which in its larval stage causes cystic hydatid disease, endemic in Southern Brazil. Epidemiological and molecular knowledge about the haplotypes occurring in a region is essential to control the spread of the disease. The aim of this work was to analyze the haplotype frequency and fertility of hydatid cysts in cattle from the state of Rio Grande do Sul. Cysts were collected and classified according to their fertility status. DNA was extracted from protoscoleces and germinal layers and then used as template for the amplification of the cytochrome c oxidase subunit 1 gene by PCR. Amplicons were purified and sequenced, and the sequences were analyzed for haplotype identification. A total of 638 fertile cysts collected in the last ten years were genotyped. On average, G1 (56.6%) was more frequent than G5 (43.4%). In lungs, the G5 haplotype exhibited a higher parasite load (52.8%), whereas in the liver, G1 was more frequent (90.4%). The analysis revealed an increase in the frequency of G5 haplotype cysts during the period of sampling, and an increase in the abundance of fertile cysts has also been observed in the last several years. Most infertile cysts were genotyped as G1. The possible factors involved in the increase in the proportion of E. ortleppi (G5) and the consequences of this increase are discussed. This study suggests that the proportion of E. ortleppi (G5) loads in cattle may be increasing overtime.

Taeniid history, natural selection and antigenic diversity: evolutionary theory meets helminthology.

Large sets of nucleotide sequence data of parasitic helminths have been accumulated in the past two decades. Our ability to improve the health of people and animals using this knowledge has not increased proportionally, however. Evolutionary biology provides the background to understand how parasites adapt to their hosts, and computational molecular biology offers the tools to infer the mechanisms involved. The study of antigenic diversity, a way for parasites to overcome host defenses against parasites, has been neglected in helminths, yet such a study could contribute to the development of more efficient drugs, diagnostic tests and vaccines. This review focuses on the study of adaptive evolution as the cause of antigenic diversity in tapeworms and its potential applications.

Cloning and partial characterization of a Boophilus microplus (Acari: Ixodidae) calreticulin.

We report the cloning, sequence characterization and expression analysis of a calreticulin (CRT) coding cDNA of Boophilus microplus. CRT is a calcium-binding protein involved in multiple cell functions and possibly implicated in parasites host immune system evasion. The CRT cDNA sequence and its molecular characterization are described. Sequence similarity and phylogenetic analyses indicate a close relationship to other arthropod CRT sequences. The CRT cDNA was also expressed in a procariotic system and the recombinant protein (rBmCRT) was used to raise antibodies in a rabbit. Expression analyses of the corresponding gene in different developmental stages and tissues were performed by RT-PCR and Western-blot, which indicated a ubiquitous expression of the B. microplus calreticulin gene and demonstrated its presence in saliva. Sera of tick-infested bovines suggested that this protein may not be able to induce an IgG-based humoral response in its natural host.

Several strains of Echinococcus granulosus infect livestock and humans in Argentina.

Mitochondrial cytochrome oxidase subunit 1 (CO1) sequencing, Southern blot of a repetitive DNA element and single strand conformation polymorphism of the 5' non-transcribed region of the cytosolic malate dehydrogenase (MDH) gene were used to determine the extent and distribution of Echinococcus granulosus genetic variation in Argentina. Five distinct strains of E. granulosus were shown to exist in the country. The common sheep, Tasmanian sheep, cattle and camel strains were identified in humans. Unlike the situation found in other countries, where the common sheep strain is the major source of human contamination, the Tasmanian sheep and camel strains produced a significant number of human infections in some regions of Argentina. This is the first report of cattle strain in humans in South America. Goats could be the natural intermediate host of the camel strain, which was not identified in humans from other regions so far. More than one genotype was identified in the same geographic area. These findings may have important consequences for human health and the control of hydatid disease. Within-strain differences were also observed, showing the potential of variation of E. granulosus.