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.

Evolution of a morphological novelty occurred before genome compaction in a lineage of extreme parasites.

Intracellular parasitism results in extreme adaptations, whose evolutionary history is difficult to understand, because the parasites and their known free-living relatives are so divergent from one another. Microsporidia are intracellular parasites of humans and other animals, which evolved highly specialized morphological structures, but also extreme physiologic and genomic simplification. They are suggested to be an early-diverging branch on the fungal tree, but comparisons to other species are difficult because their rates of molecular evolution are exceptionally high. Mitochondria in microsporidia have degenerated into organelles called mitosomes, which have lost a genome and the ability to produce ATP. Here we describe a gut parasite of the crustacean Daphnia that despite having remarkable morphological similarity to the microsporidia, has retained genomic features of its fungal ancestors. This parasite, which we name Mitosporidium daphniae gen. et sp. nov., possesses a mitochondrial genome including genes for oxidative phosphorylation, yet a spore stage with a highly specialized infection apparatus--the polar tube--uniquely known only from microsporidia. Phylogenomics places M. daphniae at the root of the microsporidia. A comparative genomic analysis suggests that the reduction in energy metabolism, a prominent feature of microsporidian evolution, was preceded by a reduction in the machinery controlling cell cycle, DNA recombination, repair, and gene expression. These data show that the morphological features unique to M. daphniae and other microsporidia were already present before the lineage evolved the extreme host metabolic dependence and loss of mitochondrial respiration for which microsporidia are well known.

Single-nucleotide polymorphisms of two closely related microsporidian parasites suggest a clonal population expansion after the last glaciation.

The mode of reproduction of microsporidian parasites has remained puzzling since many decades. It is generally accepted that microsporidia are capable of sexual reproduction, and that some species have switched to obligate asexuality, but such process had never been supported with population genetic evidence. We examine the mode of reproduction of Hamiltosporidium tvaerminnensis and Hamiltosporidium magnivora, two closely related microsporidian parasites of the widespread freshwater crustacean Daphnia magna, based on a set of 129 single-nucleotide polymorphisms distributed across 16 genes. We analyse 20 H. tvaerminnensis isolates from localities representative of the entire species' geographic distribution along the Skerry Island belt of the Baltic Sea. Five isolates of the sister species H. magnivora were used for comparison. We estimate the recombination rates in H. tvaerminnensis to be at least eight orders of magnitude lower than in H. magnivora and not significantly different from zero. This is corroborated by the higher divergence between H. tvaerminnensis alleles (including fixed heterozygosity), as compared to H. magnivora. Our study confirms that sexual recombination is present in microsporidia, that it can be lost, and that asexuals may become epidemic.

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.

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.

Distinguishing Echinococcus granulosus sensu stricto genotypes G1 and G3 with confidence: A practical guide.

Cystic echinococcosis (CE), a zoonotic disease caused by tapeworms of the species complex Echinococcus granulosus sensu lato, represents a substantial global health and economic burden. Within this complex, E. granulosus sensu stricto (genotypes G1 and G3) is the most frequent causative agent of human CE. Currently, there is no fully reliable method for assigning samples to genotypes G1 and G3, as the commonly used mitochondrial cox1 and nad1 genes are not sufficiently consistent for the identification and differentiation of these genotypes. Thus, a new genetic assay is required for the accurate assignment of G1 and G3. Here we use a large dataset of near-complete mtDNA sequences (n = 303) to reveal the extent of genetic variation of G1 and G3 on a broad geographical scale and to identify reliable informative positions for G1 and G3. Based on extensive sampling and sequencing data, we developed a new method, that is simple and cost-effective, to designate samples to genotypes G1 and G3. We found that the nad5 is the best gene in mtDNA to differentiate between G1 and G3, and developed new primers for the analysis. Our results also highlight problems related to the commonly used cox1 and nad1. To guarantee consistent identification of G1 and G3, we suggest using the sequencing of the nad5 gene region (680 bp). This region contains six informative positions within a relatively short fragment of the mtDNA, allowing the differentiation of G1 and G3 with confidence. Our method offers clear advantages over the previous ones, providing a significantly more consistent means to distinguish G1 and G3 than the commonly used cox1 and nad1.

Global phylogeography and genetic diversity of the zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1.

Echinococcus granulosus sensu stricto (s.s.) is the major cause of human cystic echinococcosis worldwide and is listed among the most severe parasitic diseases of humans. To date, numerous studies have investigated the genetic diversity and population structure of E. granulosus s.s. in various geographic regions. However, there has been no global study. Recently, using mitochondrial DNA, it was shown that E. granulosus s.s. G1 and G3 are distinct genotypes, but a larger dataset is required to confirm the distinction of these genotypes. The objectives of this study were to: (i) investigate the distinction of genotypes G1 and G3 using a large global dataset; and (ii) analyse the genetic diversity and phylogeography of genotype G1 on a global scale using near-complete mitogenome sequences. For this study, 222 globally distributed E. granulosus s.s. samples were used, of which 212 belonged to genotype G1 and 10 to G3. Using a total sequence length of 11,682 bp, we inferred phylogenetic networks for three datasets: E. granulosus s.s. (n = 222), G1 (n = 212) and human G1 samples (n = 41). In addition, the Bayesian phylogenetic and phylogeographic analyses were performed. The latter yielded several strongly supported diffusion routes of genotype G1 originating from Turkey, Tunisia and Argentina. We conclude that: (i) using a considerably larger dataset than employed previously, E. granulosus s.s. G1 and G3 are indeed distinct mitochondrial genotypes; (ii) the genetic diversity of E. granulosus s.s. G1 is high globally, with lower values in South America; and (iii) the complex phylogeographic patterns emerging from the phylogenetic and geographic analyses suggest that the current distribution of genotype G1 has been shaped by intensive animal trade.

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.

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.

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.

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.

The reduced genome of the parasitic microsporidian Enterocytozoon bieneusi lacks genes for core carbon metabolism.

Reduction of various biological processes is a hallmark of the parasitic lifestyle. Generally, the more intimate the association between parasites and hosts the stronger the parasite relies on its host's physiology for survival and reproduction. However, some systems have been held to be indispensable, for example, the core pathways of carbon metabolism that produce energy from sugars. Even the most hardened anaerobes that lack oxidative phosphorylation and the tricarboxylic acid cycle have retained glycolysis and some downstream means to generate ATP. Here we describe the deep-coverage genome resequencing of the pathogenic microsporidiian, Enterocytozoon bieneusi, which shows that this parasite has crossed this line and abandoned complete pathways for the most basic carbon metabolism. Comparing two genome sequence surveys of E. bieneusi to genomic data from four other microsporidia reveals a normal complement of 353 genes representing 30 functional pathways in E. bieneusi, except that only 2 out of 21 genes collectively involved in glycolysis, pentose phosphate, and trehalose metabolism are present. Similarly, no genes encoding proteins involved in the processing of spliceosomal introns were found. Altogether, E. bieneusi appears to have no fully functional pathway to generate ATP from glucose. Therefore, this intracellular parasite relies on transporters to import ATP from its host.

Draft genome sequence of the Daphnia pathogen Octosporea bayeri: insights into the gene content of a large microsporidian genome and a model for host-parasite interactions.

BACKGROUND: The highly compacted 2.9-Mb genome of Encephalitozoon cuniculi placed the microsporidia in the spotlight, encoding a mere 2,000 proteins and a highly reduced suite of biochemical pathways. This extreme level of reduction is not universal across the microsporidia, with genomes known to vary up to sixfold in size, suggesting that some genomes may harbor a gene content that is not as reduced as that of Enc. cuniculi. In this study, we present an in-depth survey of the large genome of Octosporea bayeri, a pathogen of Daphnia magna, with an estimated genome size of 24 Mb, in order to shed light on the organization and content of a large microsporidian genome. RESULTS: Using Illumina sequencing, 898 Mb of O. bayeri genome sequence was generated, resulting in 13.3 Mb of unique sequence. We annotated a total of 2,174 genes, of which 893 encodes proteins with assigned function. The gene density of the O. bayeri genome is very low on average, but also highly uneven, so gene-dense regions also occur. The data presented here suggest that the O. bayeri proteome is well represented in this analysis and is more complex that that of Enc. cuniculi. Functional annotation of O. bayeri proteins suggests that this species might be less biochemically dependent on its host for its metabolism than its more reduced relatives. CONCLUSIONS: The combination of the data presented here, together with the imminent annotated genome of Daphnia magna, will provide a wealth of genetic and genomic tools to study host-parasite interactions in an interesting model for pathogenesis.

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.