Overcoming research challenges: In vitro cultivation of Ameson portunus (Phylum Microsporidia).

Ameson portunus is an intracellular pathogen that infects marine crabs Portunus trituberculatus and Scylla paramamosain, causing significant economic losses. However, research into this important parasite has been limited due to the absence of an in vitro culture system. To address this challenge, we developed an in vitro cultivation model of A. portunus using RK13 cell line in this study. The fluorescent labeling assay indicated a high infection rate (∼60 %) on the first day post-infection and quantitative PCR (qPCR) detection demonstrated successful infection as early as six hours post-inoculation. Fluorescence in situ hybridization (FISH) and qPCR were used for the detection of A. portunus infected cells. The FISH probe we designed allowed detection of A. portunus in infected cells and qPCR assay provided accurate quantification of A. portunus in the samples. Transmission electron microscopy (TEM) images revealed that A. portunus could complete its entire life cycle and produce mature spores in RK13 cells. Additionally, we have identified novel life cycle characteristics during the development of A. portunus in RK 13 cells using TEM. These findings contribute to our understanding of new life cycle pathways of A. portunus. The establishment of an in vitro culture model for A. portunus is critical as it provides a valuable tool for understanding the molecular and immunological events that occur during infection. Furthermore, it will facilitate the development of effective treatment strategies for this intracellular pathogen.

Single Cell Analysis Reveals a New Microsporidia-Host Association in a Freshwater Lake.

Microsporidia are a large group of obligate intracellular eukaryotic parasites. Recent studies suggest that their diversity can be huge in freshwater lake ecosystems especially in the < 150-µm size fraction. However, little is known about their hosts and therefore their impact on the trophic food web functioning. In this study, single cell analysis and fluorescence microscopy were used to detect new host-parasite association within rotifer communities in lake Aydat (France). Our analysis showed the existence of a potential new species belonging to the Crispospora genus able of infecting the rotifer Kellicottia with a high prevalence (42.5%) suggesting that Microsporidia could have a great impact on the rotifer populations' regulation in lakes.

The immune response of Locusta migratoria manilensis at different times of infection with Paranosema locustae.

Paranosema locustae is an entomopathogenic microsporidia with promising potential for controlling agricultural pests, including Locusta migratoria manilensis. However, it has the disadvantage of having a slow insecticidal rate, and how P. locustae infection impacts the host immune response is currently unknown. The present study investigated the effect of P. locustae on the natural immune response of L. migratoria and the activities of enzymes that protect against oxidative stress. Infection with P. locustae increased the hemocytes and nodulation number of L. migratoria at the initial stage of infection. The hemocyte-mediated modulation of immune response was also affected by a decrease in the number of hemocytes 12 days postinfection. Superoxide dismutase activity in locusts increased in the early stages of infection but decreased in the later stages, whereas the activities of peroxidase (POD) and catalase (CAT) showed opposite trends may be due to their different mechanisms of action. Furthermore, the transcription levels of mRNA of antimicrobial peptide-related genes and phenoloxidase activity in hemolymph in L. migratoria were suppressed within 15 days of P. locustae infection. Overall, our data suggest that P. locustae create a conducive environment for its own proliferation in the host by disrupting the immune defense against it. These findings provide useful information for the potential application of P. locustae as a biocontrol agent.

Ubiquitin-related processes and innate immunity in C. elegans.

Innate immunity is an evolutionary ancient defence strategy that serves to eliminate infectious agents while maintaining host health. It involves a complex network of sensors, signaling proteins and immune effectors that detect the danger, then relay and execute the immune programme. Post-translational modifications relying on conserved ubiquitin and ubiquitin-like proteins are an integral part of the system. Studies using invertebrate models of infection, such as the nematode Caenorhabditis elegans, have greatly contributed to our understanding of how ubiquitin-related processes act in immune sensing, regulate immune signaling pathways, and participate to host defence responses. This review highlights the interest of working with a genetically tractable model organism and illustrates how C. elegans has been used to identify ubiquitin-dependent immune mechanisms, discover novel ubiquitin-based resistance strategies that mediate pathogen clearance, and unravel the role of ubiquitin-related processes in tolerance, preserving host fitness during pathogen attack. Special emphasis is placed on processes that are conserved in mammals.

Squash bees host high diversity and prevalence of parasites in the northeastern United States.

The squash bee Eucera (Peponapis) pruinosa is emerging as a model species to study how stressors impact solitary wild bees in North America. Here, we describe the prevalence of trypanosomes, microsporidians and mollicute bacteria in E. pruinosa and two other species, Bombus impatiens and Apis mellifera, that together comprise over 97% of the pollinator visitors of Cucurbita agroecosystems in Pennsylvania (United States). Our results indicate that all three parasite groups are commonly detected in these bee species, but E. pruinosa often exhibit higher prevalences. We further describe novel trypanosome parasites detected in E. pruinosa, however it is unknown how these parasites impact these bees. We suggest future work investigates parasite replication and infection outcomes.

The ins and outs of host-microsporidia interactions during invasion, proliferation and exit.

Microsporidia are a large group of fungal-related obligate intracellular parasites. They are responsible for infections in humans as well as in agriculturally and environmentally important animals. Although microsporidia are abundant in nature, many of the molecular mechanisms employed during infection have remained enigmatic. In this review, we highlight recent work showing how microsporidia invade, proliferate and exit from host cells. During invasion, microsporidia use spore wall and polar tube proteins to interact with host receptors and adhere to the host cell surface. In turn, the host has multiple defence mechanisms to prevent and eliminate these infections. Microsporidia encode numerous transporters and steal host nutrients to facilitate proliferation within host cells. They also encode many secreted proteins which may modulate host metabolism and inhibit host cell defence mechanisms. Spores exit the host in a non-lytic manner that is dependent on host actin and endocytic recycling proteins. Together, this work provides a fuller picture of the mechanisms that these fascinating organisms use to infect their hosts.

The metabolic theory of ecology and the cost of parasitism.

With over 1 million species on earth, each biologically unique, do we have any hope of understanding whether species will persist in a warming world? We might, because it turns out that there is surprising regularity in how warming accelerates the major metabolic processes that power life. A persistent challenge has been to understand ecological effects of temperature in the context of species interactions, especially when individuals not only experience temperature but also mortality due to parasitism or predation. Kirk et al. have shown how the effects of parasites vary with warming in a manner entirely consistent with general temperature dependence of host and parasite metabolism.

Empirical evidence that metabolic theory describes the temperature dependency of within-host parasite dynamics.

The complexity of host-parasite interactions makes it difficult to predict how host-parasite systems will respond to climate change. In particular, host and parasite traits such as survival and virulence may have distinct temperature dependencies that must be integrated into models of disease dynamics. Using experimental data from Daphnia magna and a microsporidian parasite, we fitted a mechanistic model of the within-host parasite population dynamics. Model parameters comprising host aging and mortality, as well as parasite growth, virulence, and equilibrium abundance, were specified by relationships arising from the metabolic theory of ecology. The model effectively predicts host survival, parasite growth, and the cost of infection across temperature while using less than half the parameters compared to modeling temperatures discretely. Our results serve as a proof of concept that linking simple metabolic models with a mechanistic host-parasite framework can be used to predict temperature responses of parasite population dynamics at the within-host level.

Two parasites in one host: spatiotemporal dynamics and co-occurrence of Microsporidia and Rickettsia in an amphipod host.

Biological interactions can greatly influence the abundance of species. This is also true for parasitic species that share the same host. Microsporidia and Rickettsia are widespread intracellular parasites in populations of Paracalliope fluviatilis, the most common freshwater amphipods in New Zealand. Although both parasites coexist in many populations, it is unclear whether they interact with each other. Here, we investigated spatial−temporal dynamics and co-occurrence of the two parasites, Microsporidia and Rickettsia in P. fluviatilis hosts, across one annual cycle and in three different locations. Prevalence of both Microsporidia and Rickettsia changed over time. However, while the prevalence of Rickettsia varied significantly between sampling times, that of Microsporidia did not change significantly and remained relatively low. The two parasites therefore followed different temporal patterns. Also, the prevalence of both parasites differed among locations, though the two species reached their highest prevalence in different locations. Lastly, there was no evidence for positive or negative associations between the two parasite species; the presence of one parasite in an individual host does not appear to influence the probability of infection by the other parasite. Their respective prevalence may follow different patterns among populations on a larger spatial scale due to environmental heterogeneity across locations.

Molecular phylogeny and new light microscopic data of Metchnikovella spiralis (Microsporidia: Metchnikovellidae), a hyperparasite of eugregarine Polyrhabdina sp. from the polychaete Pygospio elegans.

Metchnikovellids are a deep-branching group of microsporidia, parasites of gregarines inhabiting the alimentary tract of polychaetes and some other invertebrates. The diversity and phylogeny of these hyperparasites remain poorly studied. Modern descriptions and molecular data are still lacking for many species. The results of a light microscopy study and molecular data for Metchnikovella spiralis Sokolova et al., 2014, a hyperparasite of the eugregarine Polyrhabdina sp., isolated from the polychaete Pygospio elegans, were obtained. The original description of M. spiralis was based primarily on the analysis of stained preparations and transmission electron microscopy images. Here, the species description was complemented with the results of in vivo observations and phylogenetic analysis based on the SSU rRNA gene. It was shown that in this species, free sporogony precedes sac-bound sporogony, as it occurs in the life cycle of most other metchnikovellids. Spore sacs are entwined with spirally wound cords, and possess only one polar plug. Phylogenetic analyses did not group M. spiralis with M. incurvata, another metchnikovellid from the same gregarine species, but placed it as a sister branch to Amphiacantha. The paraphyletic nature of the genus Metchnikovella was discussed. The taxonomic summary for M. spiralis was emended.

Microsporidia: Eukaryotic Intracellular Parasites Shaped by Gene Loss and Horizontal Gene Transfers.

Microsporidia are eukaryotic parasites of many animals that appear to have adapted to an obligate intracellular lifestyle by modifying the morphology and content of their cells. Living inside other cells, they have lost many, or all, metabolic functions, resulting in genomes that are always gene poor and often very small. The minute content of microsporidian genomes led many to assume that these parasites are biochemically static and uninteresting. However, recent studies have demonstrated that these organisms can be surprisingly complex and dynamic. In this review I detail the most significant recent advances in microsporidian genomics and discuss how these have affected our understanding of many biological aspects of these peculiar eukaryotic intracellular pathogens.

Microsporidia Can Acquire Lamin-like Intermediate Filaments and Cell Adhesion Catenin-cadherin Complexes from the Host (?).

On their spore surfaces, Microsporidia often develop a canopy of filaments with characteristics of intermediate filaments (IF), as we demonstrated in previous studies on Thelohania sp., Ameson michaelis, and Spraguea lophii. Genomic studies indicate that among invertebrates, lamins that may localize in the cytoplasm or nucleus, are the only known IF type. These IFs can bind to the substrate containing cell adhesion molecules (CAMs) cadherins, associated with ß and γ catenins. The objects of this study were to determine whether microsporidia have CAMs with the attached IFs on their envelopes and to find out if these proteins are provided by the host. An examination was made for localization of lamins and CAMs on the spores of the mentioned above species and Anncaliia algerae, plus in the host animals. Then, we determined whether the spores of A. michaelis and A. algerae could bind vertebrate nuclear lamin onto the spore surface. We also tested transgenic Drosophila melanogaster stocks bearing cadherin-GFP to see whether developing A. algerae parasites in these hosts could acquire host CAMs. The tests were positive for all these experiments. We hypothesize that microsporidia are able to acquire host lamin IFs and cell adhesion catenin-cadherin complexes from the host.

Acid and heat fastness in microsporidia: How acid fast are acid fast microsporidium?

Microsporidia are obligate spore-forming microorganisms with strong resemblance to fungi and can affect almost every organ system in immunocompetent or immunocompromised individuals. Mixed infections are also reported in immunocompromised hosts. Microsporidial spores show marked morphological variations and the small and slender forms can resemble bacilli. Modified Zeihl Neelsen (ZN) stain, cold method demonstrates them as bright red in color, leaving several spores blue or incompletely stained; thus, they are reported as weakly or variably acid fast. Variability in staining results with ZN stain and considering the fact that Mycobacterium tuberculosis, the commoner bug in developing countries is identified by its resistance to stronger acids on ZN staining, authors wished to demonstrate acid and heat fastness in microsporidium using corneal tissue specimens. Microsporidial spores stained bright red in color with conventional ZN stain, demonstrated strong acid fastness, and interestingly the staining results improved on heating. Thus, the authors conclude that they are strongly acid and heat fast and care must be warranted so that they are not misdiagnosed as Mycobacterium or other acid-fast organisms. Careful observation of morphology, battery of special stains, and molecular diagnostics should be advocated for diagnostic confirmation. To the best of the authors' knowledge, this is the first explicit report on acid and heat fastness on microsporidial spores.

Ovipleistophora diplostomuri, a parasite of fish and their trematodes, also infects the crayfish Procambarus bivittatus.

Ovipleistophora diplostomuri (Microsporidia) is an obligate parasite of fish and trematodes in the US. In April 2019, an individual crayfish, Procambarus bivittatus (Escambia River, Florida), with a high-intensity microsporidian infection was delivered to the Emerging Pathogens Institute. Histological analysis determined that infection was restricted to the muscle tissue. Molecular diagnostics (PCR) provided 952 bp of the parasite SSU (18S) sequence. The isolate was 99.16% similar to O. diplostomuri identified from blue gill and their trematode parasites in Washington, USA. This discovery increases our understanding of Microsporidia within aquatic trophic networks, supporting the theory that the Ovipleistophora share complex relationships with vertebrates, invertebrates and helminth parasites.

Geographic distribution of Ovavesicula popilliae in the United States and sensitivity of visual diagnosis compared with qPCR detection.

The Japanese beetle (Popillia japonica) is one of the most destructive invasive pests in North America, causing significant economic impact to many fruit crops, turfgrass and the nursery industry. A microsporidian pathogen of Japanese beetle, Ovavesicula popilliae, discovered in 1985, proliferates in the Malpighian tubules of larvae and adults, disrupting waste-removal, mineral filtering, and fluid balance in heavily infected individuals. Most infected larvae do not survive from fall to spring, and egg production by infected females is reduced by 50%. Ovavesicula popilliae is promising as a classical biological control agent for Japanese beetle, but outside of surveys completed in Connecticut and Michigan little is known about its geographic distribution in North America. The objective of this research is to obtain a better understanding of the distribution of O. popilliae in North America. Japanese beetles were collected at 59 locations in a total of 19 different states in the USA for pathogen analysis. Overall, the proportion of Japanese beetle adults infected by O. popilliae was much greater in Michigan, Ohio, Tennessee and four states in the northeastern USA compared with sites located west of the Mississippi River (18.6 ± 13.3% and 0.6 ± 1.2%, respectively). Nucleotide sequences of the gene encoding a small subunit of nuclear ribosomal RNA (ssrDNA), obtained from GenBank for O. popilliae was used to develop a highly specific qPCR test for O. popilliae DNA. A subsample of 110 individual Japanese beetles were visually diagnosed first, then analyzed via qPCR. Visual diagnosis and qPCR detection agreed for 80.9% of the beetles tested. The qPCR assay is more sensitive than visual diagnosis (56 visually positive, 73 qPCR positive), is highly specific for O. popilliae, and will be useful for detecting the pathogen in large batches of beetles, or in beetle frass.

Infectivity of Paranosema locustae (Microsporidia) against gregarious-phase South American locust (Orthoptera) when treated en masse.

En masse inoculations with Paranosema locustae, an intracellular parasite of adipose tissue of grasshoppers and locusts and the only microsporidium registered as a biocontrol agent, were conducted against crowded fourth-instar nymphs of the South American locust Schistocerca cancellata and the grasshoppers Dichroplus schulzi and Ronderosia bergii. Infection did not develop in the locust, but was highly prevalent in the two grasshopper species. We hypothesize that absolute absence of infection in S. cancellata may constitute a case of density-dependent prophylactic resistance, an elevation of the baseline immunity of an organism in order to cope with disease that is prevalent in species exhibiting phase polyphenism.

The diversity of microsporidian parasites infecting the Holarctic amphipod Gammarus lacustris from the Baikal region is dominated by the genus Dictyocoela.

Microsporidia are a highly diverse group of single-celled eukaryotic parasites related to fungi and infecting hosts belonging to all groups of eukaryotes, including some protists, invertebrate and vertebrate animals. We investigated the diversity of microsporidia in the Holarctic amphipod species Gammarus lacustris from mostly, but not limited to, water bodies in the Lake Baikal region. Ribosomal DNA sequencing and host transcriptome sequencing data from various works show that this species is predominantly infected by representatives of the genus Dictyocoela and probably has some features underlying this specific interaction.

Survival of Japanese beetle, Popillia japonica Newman, larvae in field plots when infected with a microsporidian pathogen, Ovavesicula popilliae.

Japanese beetle, Popillia japonica Newman, is an important invasive pest that causes significant damage to golf courses, blueberries, raspberries, hops and many other crops and ornamentals in the eastern United States. This study was conducted to determine the survival of Ovavesicula popilliae-infected larvae compared with uninfected larvae from October to May. Larvae were collected from two sites, one where O. popilliae was active and one where it had not yet been detected. Larvae were placed into plastic sleeve-pots containing 15 cm-diameter cores of turfgrass with roots and soil intact. Larvae collected from both locations were put into sleeve-pots at both locations to account for soil and site factors. Results of this experiment in both years confirm that Japanese beetle larvae infected with O. popilliae do not survive well from October to May. We estimate that at an epizootic location where the pathogen has been active for several years, at least 76.5% of the Japanese beetle larvae infected in October do not survive until May. When the observed amount of population reduction (27-29%) due to natural pathogen infection of larvae in our field plots is combined with a 50% reduction in eggs produced by infected females as previously reported, annual population declines due to O. popilliae would average 40% (assuming a typical adult female infection rate of 25%). This rate of population reduction is consistent with previous reports of Japanese beetle population decline over a period of several years at O. popilliae epizootic sites.

Characterization of Hsp70 gene family provides insight into its functions related to microsporidian proliferation.

Heat shock protein 70 (Hsp70), a highly conserved protein family, is widely distributed in organisms and plays fundamental roles in biotic and abiotic stress responses. However, reports on Hsp70 genes are scarce in microsporidia, a very large group of obligate intracellular parasites that can infect nearly all animals, including humans. In this study, we identified 37 Hsp70 proteins from eight microsporidian genomes and classified them into four subfamilies (A-D). The number of Hsp70 genes in these microsporidia was significantly fewer than in Rozella allomycis and yeast. All microsporidian species contained genes from each subfamily and similar subcellular locations (mitochondria, endoplasmic reticulum, cytosol, and cytosol and/or nucleus), indicating that each Hsp70 member may fulfil distinct functions. The conserved structures and motifs of the Hsp70 proteins in the same subfamily were highly similar. Expression analysis indicated that the subfamily C cytosol (cyto)-associated Hsp70s is functional during microsporidia development. Immunofluorescence assays revealed that Cyto-NbHsp70 was cytoplasmically located in the proliferation-stage of Nosema bombycis. Cyto-NbHsp70 antiserum also labeled Encephalitozoon hellem within infected cells, suggesting that this antiserum is a potential molecular marker for labeling the proliferative phases of different microsporidian species. The propagation of N. bombycis was significantly inhibited following RNAi of Cyto-NbHsp70, indicating that Cyto-NbHsp70 is important for pathogen proliferation. Our phylogenetic data suggest that Hsp70 proteins evolved during microsporidia adaption to intracellular parasitism, and they play important roles in pathogen development.

Effects of Pseudoloma neurophilia infection on the brain transcriptome in zebrafish (Danio rerio).

Laboratory zebrafish are commonly infected with the intracellular, brain-infecting microsporidian parasite Pseudoloma neurophilia. Chronic P. neurophilia infections induce inflammation in meninges, brain and spinal cord, and have been suggested to affect neural functions since parasite clusters reside inside neurons. However, underlying neural and immunological mechanisms associated with infection have not been explored. Utilizing RNA-sequencing analysis, we found that P. neurophilia infection upregulated 175 and downregulated 45 genes in the zebrafish brain, compared to uninfected controls. Four biological pathways were enriched by the parasite, all of which were associated with immune function. In addition, 14 gene ontology (GO) terms were enriched, eight of which were associated with immune responses and five with circadian rhythm. Surprisingly, no differentially expressed genes or enriched pathways were specific for nervous system function. Upregulated immune-related genes indicate that the host generally show a pro-inflammatory immune response to infection. On the other hand, we found a general downregulation of immune response genes associated with anti-pathogen functions, suggesting an immune evasion strategy by the parasite. The results reported here provide important information on host-parasite interaction and highlight possible pathways for complex effects of parasite infections on zebrafish phenotypes.