Microsporidian Species Research Articles

Microsporidians (Microsporidia) parasitic on mosquitoes (Culicidae) in central Europe are often multi-host species

Microsporidians (Microsporidia) are a diverse group of obligate and intracellular parasites of eukaryotes. There is evidence that the real species diversity in the phylum could be greatly underestimated, especially for microsporidians parasitic on invertebrates. Mosquitoes (Culicidae) are among very important microsporidian host groups. However, to date, no extensive survey on the prevalence of microsporidians in European mosquitoes has been performed. Here, we used mosquitoes collected in west-central Poland and a metabarcoding approach to examine the prevalence and diversity of microsporidian species among European mosquitoes. We found that up to one-third of mosquitoes in Europe may be infected with at least 13 microsporidian species belonging to the genera Amblyospora, Hazardia, Encephalitozoon, Enterocytospora, and Nosema and the holding genus Microsporidium. The lack of a difference in microsporidian prevalence between mosquito sexes implies that other factors, e.g., temperature or humidity, affect microsporidian occurrence in adult mosquitoes. Each microsporidian species was found in at least three mosquito species, which suggests that these microsporidians are polyxenic rather than monoxenic parasites. The co-occurrence of at least two different microsporidian species was found in 3.6% of host individuals. The abundance of microsporidian DNA sequences suggests interactions between co-occurring parasites; however, these results should be confirmed by microscopic and quantitative methods. In addition, further histological research is required to describe Microsporidium sp. PL01 or match its DNA to that of an already described species.

Inhibition of mitosomal alternative oxidase causes lifecycle arrest of early-stage Trachipleistophora hominis meronts during intracellular infection of mammalian cells.

Mitosomes are highly reduced forms of mitochondria which have lost two of the 'defining' features of the canonical organelle, the mitochondrial genome, and the capacity to generate energy in the form of ATP. Mitosomes are found in anaerobic protists and obligate parasites and, in most of the studied organisms, have a conserved function in the biosynthesis of iron-sulfur clusters (ISC) that are indispensable cofactors of many essential proteins. The genomes of some mitosome-bearing human pathogenic Microsporidia encode homologues of an alternative oxidase (AOX). This mitochondrial terminal respiratory oxidase is absent from the human host, and hence is a potential target for the development of new antimicrobial agents. Here we present experimental evidence for the mitosomal localization of AOX in the microsporidian Trachipleistophora hominis and demonstrate that it has an important role during the parasite's life cycle progression. Using a recently published methodology for synchronising T. hominis infection of mammalian cell lines, we demonstrated specific inhibition of T. hominis early meront growth and replication by an AOX inhibitor colletochlorin B. Treatment of T. hominis-infected host cells with the drug also inhibited re-infection by newly formed dispersive spores. Addition of the drug during the later stages of the parasite life cycle, when our methods suggest that AOX is not actively produced and T. hominis mitosomes are mainly active in Fe/S cluster biosynthesis, had no inhibitory effects on the parasites. Control experiments with the AOX-deficient microsporidian species Encephalitozoon cuniculi, further demonstrated the specificity of inhibition by the drug. Using the same methodology, we demonstrate effects of two clinically used anti-microsporidian drugs albendazole and fumagillin on the cell biology and life cycle progression of T. hominis infecting mammalian host cells. In summary, our results reveal that T. hominis mitosomes have an active role to play in the progression of the parasite life cycle as well as an important role in the biosynthesis of essential Fe/S clusters. Our work also demonstrates that T. hominis is a useful model for testing the efficacy of therapeutic agents and for studying the physiology and cell biology of microsporidian parasites growing inside infected mammalian cells.

Conservation of Nematocida microsporidia gene expression and host response in Caenorhabditis nematodes.

Microsporidia are obligate intracellular parasites that are known to infect most types of animals. Many species of microsporidia can infect multiple related hosts, but it is not known if microsporidia express different genes depending upon which host species is infected or if the host response to infection is specific to each microsporidia species. To address these questions, we took advantage of two species of Nematocida microsporidia, N. parisii and N. ausubeli, that infect two species of Caenorhabditis nematodes, C. elegans and C. briggsae. We performed RNA-seq at several time points for each host infected with either microsporidia species. We observed that Nematocida transcription was largely independent of its host. We also observed that the host transcriptional response was similar when infected with either microsporidia species. Finally, we analyzed if the host response to microsporidia infection was conserved across host species. We observed that although many of the genes upregulated in response to infection are not direct orthologs, the same expanded gene families are upregulated in both Caenorhabditis hosts. Together our results describe the transcriptional interactions of Nematocida infection in Caenorhabditis hosts and demonstrate that these responses are evolutionarily conserved.

Molecular Detection of Microsporidia in Rabbits (Oryctolagus cuniculus) in Tenerife, Canary Islands, Spain.

Enterocytozoon bieneusi and Encephalitozoon spp. are microsporidia with zoonotic potential that have been identified in humans, as well as in a large group of wild and domestic animals. Several wildlife species have been studied as reservoirs of zoonotic microsporidia in mainland Spain, including the European rabbit (Oryctolagus cuniculus). Due to a lack of data on microsporidia infection in wildlife on the Canary Islands, the aim of this work was to analyze the prevalence and identify the species of microsporidia in rabbits in Tenerife. Between 2015 and 2017, a total of 50 fecal samples were collected from rabbits in eight municipalities of Tenerife, Canary Islands, Spain. Seven of the fifty samples (14%) were amplified using nested polymerase chain reaction (PCR) targeting the partial sequence of the 16S rRNA gene, the internal transcribed spacer (ITS) region, and the partial sequence of the 5.8S rRNA gene. Sanger sequencing reveals the presence of Encephalitozoon cuniculi genotype I in two samples (4%), and undescribed microsporidia species in five samples (10%). This study constitutes the first molecular detection and genotyping of E. cuniculi in rabbits in Spain.

Characterizing the Proliferation Patterns of Representative Microsporidian Species Enlightens Future Studies of Infection Mechanisms.

Microsporidia are a group of pathogens that infect all kinds of animals, such as humans, silkworms, honeybees, and shrimp; they, therefore, pose a severe threat to public health and the economy. There are over 1500 species of microsporidia that have been reported, among which Encephalitozoon hellem and Nosema bombycis are the representative zoonotic and insect-infecting species, respectively. Investigating their cell infection patterns is of great significance for understanding their infection mechanisms. Specific probes were designed for the ribosomal RNA sequences of microsporidia. Fluorescence in situ hybridization (FISH) was used to trace the proliferation cycle of the pathogens in different cells. Here, two rRNA large subunit gene (LSUrRNA) probes specifically labeling N. bombycis were obtained. The life cycle of N. bombycis in silkworm cells and E. hellem in three kinds of host cells was graphically drawn. N. bombycis meronts were first observed at 30 hours post-infection (hpi), and they began merogony. Sporonts were observed at 42 hpi, and the first entire proliferation cycle was completed at 48 hpi. The proliferation cycle of E. hellem in RK13 and HEK293 epithelial cells was almost the same, completing the first life cycle after 24 hpi, but it was significantly delayed to 32 hpi in RAW264.7. Specific FISH probes were established for labeling microsporidia in multiple host cells. The proliferation characteristics of representative zoonotic and insect-infecting microsporidian species were clarified. This study provides an experimental pattern for future analyses of microsporidian infection mechanisms.

Human Microsporidiosis: A Narrative Review of Serodiagnostic Tests

Context: Microsporidiosis is an emerging disease in both immunocompetent and immunocompromised patients. Diagnosis of human microsporidiosis is challenging. Parasitological tests using staining methods have limitations for detecting spores in stool samples. The performance of molecular methods for diagnosing human microsporidiosis is satisfactory, but these methods are expensive and time-consuming. Serodiagnosis tests for the detection of antibodies have insufficient specificity. Detecting microsporidia antigens seems to be an appropriate method for diagnosing human microsporidiosis. Therefore, the present study reviewed serological approaches to assess the current status of these methods for diagnosing human microsporidiosis. Evidence Acquisition: All related published articles were searched. The search terms were “microsporidiosis”, “microsporidiasis”, “human microsporidiosis”, and “human microsporidiasis”, combined with the search terms “diagnosis”, “serodiagnosis”, “immunodiagnosis”, “antigen detection”, and “antibody detection”. Data were extracted from the articles that met our eligibility criteria. Immunodiagnosis studies in experimentally infected animals were excluded. Results: Few studies have evaluated the performance of serological tests for diagnosing human microsporidiosis. The IgG response against spore wall protein or polar tube antigens remains at least three years after infection and shows more cross-reactivity with other species. Therefore, the western blot should be conducted as a confirmatory method. Serodiagnosis tests using monoclonal antibodies against microsporidia showed less cross-reactivity and more efficacy compared to polyclonal antibodies. Conclusions: Indirect fluorescent antibody test based on the monoclonal antibody is highly effective in diagnosing microsporidia species in clinical samples.

High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans.

Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia represent to humans and agricultural animals, few reliable treatment options exist. Here, we develop a high-throughput screening method for the identification of chemical inhibitors of microsporidia infection, using liquid cultures of Caenorhabditis elegans infected with the microsporidia species Nematocida parisii. We screen a collection of 2560 FDA-approved compounds and natural products, and identify 11 candidate microsporidia inhibitors. Five compounds prevent microsporidia infection by inhibiting spore firing, whereas one compound, dexrazoxane, slows infection progression. The compounds have in vitro activity against several other microsporidia species, including those known to infect humans. Together, our results highlight the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens, and provide a scalable high-throughput system for the identification and characterization of microsporidia inhibitors.

Microsporidia as a Potential Threat to the Iberian Lynx (Lynx pardinus).

Simple SummaryThe Iberian lynx, which inhabits the Iberian Peninsula, is one of the most endangered felines in the world. Wild Iberian lynx populations have suffered a constant regression over the past century, with a rapid decline of 90% in the last 20 years. Infectious diseases are one of the most critical threats that cause the population decline of these animals, either in the wild or captivity. Different studies have revealed positive seroprevalence against various pathogens, confirming contact and exposure to bacteria, viruses, and parasites. In this sense, searching for pathogens related to the depopulation of the Iberian lynx is vital for conserving and maintaining this threatened species. The present work confirmed the presence of microsporidia, opportunistic intracellular parasites recently related to fungi, in the lynx environment. Also, different species of microsporidia were determined for the first time in the urine, feces, and tissue samples of Lynx pardinus. Further studies are needed to establish the impact of microsporidia infection on the survival of the Iberian lynx. These studies would contribute to the endurance and conservation of this feline by implementing new prevention strategies.Lynx pardinus is one of the world’s most endangered felines inhabiting the Iberian Peninsula. The present study was performed to identify the presence of microsporidia due to the mortality increase in lynxes. Samples of urine (n = 124), feces (n = 52), and tissues [spleen (n = 13), brain (n = 9), liver (n = 11), and kidney (n = 10)] from 140 lynxes were studied. The determination of microsporidia was evaluated using Weber’s chromotrope stain and Real Time-PCR. Of the lynxes analyzed, stains showed 10.48% and 50% positivity in urine and feces samples, respectively. PCR confirmed that 7.69% and 65.38% belonged to microsporidia species. The imprints of the tissues showed positive results in the spleen (38.46%), brain (22.22%), and liver (27.27%), but negative results in the kidneys. PCR confirmed positive microsporidia results in 61.53%, 55.55%, 45.45%, and 50%, respectively. Seroprevalence against Encephalitozoon cuniculi was also studied in 138 serum samples with a positivity of 55.8%. For the first time, the results presented different species of microsporidia in the urine, feces, and tissue samples of Lynx pardinus. The high titers of anti-E. cuniculi antibodies in lynx sera confirmed the presence of microsporidia in the lynx environment. New studies are needed to establish the impact of microsporidia infection on the survival of the Iberian lynx.

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.

Zoonotic Enterocytozoon bieneusi genotypes in free-ranging and farmed wild ungulates in Spain.

Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n=1058) and farmed (n=324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animals.

Molecular detection and characterization of Enterocytozoon bieneusi in captive alpine musk deer (Moschus chrysogaster) in Central and Eastern of Gansu Province, China.

The most ubiquitously diagnosed microsporidian species in animals and humans is Enterocytozoon bieneusi (E. bieneusi). In this case, this work aimed to probe the occurrence and genotypes of this pathogen in captive alpine musk deer (Moschus chrysogaster) in Gansu Province, China. After fecal sample collection (n = 201) from three farms in Gansu, the internal transcribed spacer (ITS) region of the rRNA gene was probed by PCR for detection and genotyping of E. bieneusi. The infection rate of E. bieneusi in alpine musk deer was 6.0% (12/201), with 0% (0/36), 7.7% (5/65) and 7.0% (7/100) in farms 1 to 3, respectively. The infection rate of E. bieneusi in young alpine musk deer (3.2%; 1/31) is lower than that of adults (6.5%; 11/170), with no evident significant differences between age groups (P > 0.05). Three known genotypes D (n = 8), EbpA (n = 3) and BEB6 (n = 1) were identified by sequence analysis. This is the first such scrutiny of E. bieneusi infection in alpine musk deer in China as per our knowledge. Genotypes D and EbpA were common in humans and animals that is suggestive of the plausible zoonotic role in E. bieneusi transmission by alpine musk deer.

Use of Thymol in Nosema ceranae Control and Health Improvement of Infected Honey Bees.

Simple SummaryIn the European Union, there is no registered product for the control of the honey bee endoparasite Nosema ceranae. Thus, researchers are looking for options for Nosema treatment. The aim of this study was to investigate the effect of a natural essential-oil ingredient (thymol) derived from Thymus vulgaris on honey bees infected with N. ceranae. Thymol exerted certain positive effects (increasing bee survival, immunity, and antioxidative protection), as well as positively affecting the spore loads in Nosema-infected bees. However, when applied to Nosema-free bees, thymol caused certain health disorders; therefore, beekeepers should be careful with its use.Nosema ceranae is the most widespread microsporidian species which infects the honey bees of Apis mellifera by causing the weakening of their colonies and a decline in their productive and reproductive capacities. The only registered product for its control is the antibiotic fumagillin; however, in the European Union, there is no formulation registered for use in beekeeping. Thymol (3-hydroxy-p-cymene) is a natural essential-oil ingredient derived from Thymus vulgaris, which has been used in Varroa control for decades. The aim of this study was to investigate the effect of thymol supplementation on the expression of immune-related genes and the parameters of oxidative stress and bee survival, as well as spore loads in bees infected with the microsporidian parasite N. ceranae. The results reveal mostly positive effects of thymol on health (increasing levels of immune-related genes and values of oxidative stress parameters, and decreasing Nosema spore loads) when applied to Nosema-infected bees. Moreover, supplementation with thymol did not induce negative effects in Nosema-infected bees. However, our results indicate that in Nosema-free bees, thymol itself could cause certain disorders (affecting bee survival, decreasing oxidative capacity, and downregulation of some immune-related gene expressions), showing that one should be careful with preventive, uncontrolled, and excessive use of thymol. Thus, further research is needed to reveal the effect of this phytogenic supplement on the immunity of uninfected bees.

Microsporidia, a Highly Adaptive Organism and Its Host Expansion to Humans

Emerging infectious disease has become the center of attention since the outbreak of COVID-19. For the coronavirus, bats are suspected to be the origin of the pandemic. Consequently, the spotlight has fallen on zoonotic diseases, and the focus now expands to organisms other than viruses. Microsporidia is a single-cell organism that can infect a wide range of hosts such as insects, mammals, and humans. Its pathogenicity differs among species, and host immunological status plays an important role in infectivity and disease severity. Disseminated disease from microsporidiosis can be fatal, especially among patients with a defective immune system. Recently, there were two Trachipleistophora hominis, a microsporidia species which can survive in insects, case reports in Thailand, one patient had disseminated microsporidiosis. This review gathered data of disseminated microsporidiosis and T. hominis infections in humans covering the biological and clinical aspects. There was a total of 22 cases of disseminated microsporidiosis reports worldwide. Ten microsporidia species were identified. Maximum likelihood tree results showed some possible correlations with zoonotic transmissions. For T. hominis, there are currently eight case reports in humans, seven of which had Human Immunodeficiency Virus (HIV) infection. It is observed that risks are higher for the immunocompromised to acquire such infections, however, future studies should look into the entire life cycle, to identify the route of transmission and establish preventive measures, especially among the high-risk groups.

Occurrence and molecular characterization of Enterocytozoon bieneusi in water buffaloes (Bubalus bubalis) in Turkey

Microsporidia are obligate intracellular fungus-like parasites that infect humans and animals worldwide. However, there is limited epidemiological data on the occurrence and molecular diversity of microsporidia in buffaloes worldwide. In the present study, fecal samples of 300 water buffaloes (Bubalus bubalis) in Kayseri, Sivas, and Samsun provinces of Turkey were investigated using two nested PCR assays targeting the rRNA of E. bieneusi and Encephalitozoon spp. All the fecal samples from water buffalo were found to be negative for Encephalitozoon spp. PCR positive isolates of E. bieneusi were bidirectionally sequenced for genotyping and phylogenetic analyses. Enterocytozoon bieneusi was the only microsporidian species identified in 8 water buffaloes with an overall molecular prevalence of 2.7%. Two known genotypes, YNDCEB-90 (n = 5) and J (n = 3) were identified by ITS sequence analysis. The YNDCEB-90 and J genotypes fall into zoonotic Group 1 and 2 of E. bieneusi in the phylogenetic tree, respectively. These findings suggested that water buffalo in Turkey are harbouring zoonotic genotypes of E. bieneusi and may have a significant risk for zoonotic transmission to humans. This is the first report of detecting E. bieneusi genotypes J and YNDCEB-90 in water buffaloes. Further insight into the epidemiology of E. bieneusi in water buffaloes in different geographical areas in Turkey will be highly important to have determined the public health significance of this pathogen.

Molecular Epidemiology and Genetic Diversity of Enterocytozoon bieneusi in Cervids from Milu Park in Beijing, China.

Simple SummaryEnterocytozoon bieneusi is the most prevalent microsporidian species that can cause significant intestinal diseases in both humans and animals worldwide. This study investigated the overall E. bieneusi prevalence of 21.9% (47/215) in captive and free-ranging deer species in Beijing, China. Thirteen E. bieneusi genotypes including six known and seven novel genotypes were identified. These resources will provide the insights to understand the veterinary and public health and the transmission dynamics between animal environments and human ones.Enterocytozoon bieneusi is the most prevalent microsporidian species that can cause zoonotic diseases in humans and animals. Despite receiving increasing attention in relation to domestic animals, there has been limited information on the infection burden of E. bieneusi in cervids. Altogether, 215 fecal samples collected from four deer species in Beijing, China were examined by nested- Polymerase Chain Reaction (PCR)targeting the internal transcribed spacer (ITS) region. The overall prevalence of E. bieneusi in deer was 21.9% (47/215), with 30.0% (24/80) in Pere David’s deer, 27.3% (15/55) in fallow deer, 12.5% (5/40) in sika deer, and 7.5% (3/40) in Chinese water deer. Thirteen E. bieneusi genotypes were identified, including six known (HLJD-V, MWC_d1, BEB6, CGC2, JLD-XV, and HND-I) and seven novel genotypes (BJED-I to BJED-V, BJFD, and BJCWD). A phylogenetic analysis showed that 38.3% of the isolates belonged to zoonotic Group 1. In addition, E. bieneusi infection was first detected in fallow deer and Chinese water deer, which could act as potential zoonotic reservoirs. Our findings suggest that E. bieneusi circulates in deer and might be of importance to public health.

Paromomycin Reduces Vairimorpha (Nosema)ceranae Infection in Honey Bees but Perturbs Microbiome Levels and Midgut Cell Function.

Paromomycin is a naturally occurring aminoglycoside antibiotic that has effects on both prokaryotic and eukaryotic microbes. However, previous reports have indicated that it has little effect on microsporidia, including Vairimorpha (Nosema) ceranae, in cell culture models. V. ceranae is one of a number of microsporidia species that cause disease in honey bees and substantial efforts to find new treatment strategies for bees that are infected with these pathogens are ongoing. When testing compounds for potential activity against V. ceranae in whole organisms, we found that paromomycin reduces the infection intensity of this parasite. Critically, the necessary doses of paromomycin have high activity against the bacteria of the honey bee microbiome and cause evident stress in bees. Microsporidia have been shown to lack an essential binding site on the ribosome that is known to allow for maximal inhibition by paromomycin. Thus, it is possible that paromomycin impacts parasite levels through non-cell autonomous effects on microsporidia infection levels via effects on the microbiome or midgut cellular function. As paromomycin treatment could cause widespread honey bee health issues in agricultural settings, it does not represent an appropriate anti-microsporidia agent for use in the field.

Protists in the Insect Rearing Industry: Benign Passengers or Potential Risk?

Simple SummaryAs human populations grow and the climate crisis deepens, humans will need to look to alternative sustainable sources of protein. The insect rearing industry is now rapidly growing to generate more sustainable sources of food and feed, and, as it does so, there will be an urgent need to better understand the role that microorganisms play in both maintaining insect health and generating disease. Protists are microbes that are neither viral, bacterial nor fungal and, therefore, are sometimes overlooked when considering microbial fauna. In this paper, we review the literature on protists that have been uncovered within insects that are being considered for rearing as food and feed. We discuss what is known about how they interact with hosts, how they may affect industrially reared insects in the future and which tools now need to be developed to better study them.As the insects for food and feed industry grows, a new understanding of the industrially reared insect microbiome is needed to better comprehend the role that it plays in both maintaining insect health and generating disease. While many microbiome projects focus on bacteria, fungi or viruses, protists (including microsporidia) can also make up an important part of these assemblages. Past experiences with intensive invertebrate rearing indicate that these parasites, whilst often benign, can rapidly sweep through populations, causing extensive damage. Here, we review the diversity of microsporidia and protist species that are found in reared insect hosts and describe the current understanding of their host spectra, life cycles and the nature of their interactions with hosts. Major entomopathogenic parasite groups with the potential to infect insects currently being reared for food and feed include the Amoebozoa, Apicomplexa, Ciliates, Chlorophyta, Euglenozoa, Ichtyosporea and Microsporidia. However, key gaps exist in the understanding of how many of these entomopathogens affect host biology. In addition, for many of them, there are very limited or even no molecular data, preventing the implementation of molecular detection methods. There is now a pressing need to develop and use novel molecular tools, coupled with standard molecular diagnostic methods, to help unlock their biology and predict the effects of these poorly studied protist parasites in intensive insect rearing systems.

Identification, expression and subcellular localization of Orc1 in the microsporidian Nosema bombycis

As a typical species of microsporidium, Nosema bombycis is the pathogen causing the pébrine disease of silkworm. Rapid proliferation of N. bombycis in host cells requires replication of genetic material. As eukaryotic origin recognition protein, origin recognition complex (ORC) plays an important role in regulating DNA replication, and Orc1 is a key subunit of the origin recognition complex. In this study, we identified the Orc1 in the microsporidian N. bombycis (NbOrc1) for the first time. The NbOrc1 gene contains a complete ORF of 987 bp in length that encodes a 328 amino acid polypeptide. Indirect immunofluorescence results showed that NbOrc1 were colocalized with Nbactin and NbSAS-6 in the nuclei of N. bombycis. Subsequently, we further identified the interaction between the NbOrc1 and Nbactin by CO-IP and Western blot. These results imply that Orc1 may be involved in the proliferation of the microsporidian N. bombycis through interacting with actin.

The Rad9-Rad1-Hus1 DNA Repair Clamp is Found in Microsporidia.

DNA repair is an important component of genome integrity and organisms with reduced repair capabilities tend to accumulate mutations at elevated rates. Microsporidia are intracellular parasites exhibiting high levels of genetic divergence postulated to originate from the lack of several proteins, including the heterotrimeric Rad9–Rad1–Hus1 DNA repair clamp. Microsporidian species from the Encephalitozoonidae have undergone severe streamlining with small genomes coding for about 2,000 proteins. The highly divergent sequences found in Microsporidia render functional inferences difficult such that roughly half of these 2,000 proteins have no known function. Using a structural homology-based annotation approach combining protein structure prediction and tridimensional similarity searches, we found that the Rad9–Rad1–Hus1 DNA clamp is present in Microsporidia, together with many other components of the DNA repair machinery previously thought to be missing from these organisms. Altogether, our results indicate that the DNA repair machinery is present and likely functional in Microsporidia.

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.