Phylogeny, morphology, virulence, ecology, and host range of Ordospora pajunii (Ordosporidae), a microsporidian symbiont of Daphnia spp.

The impacts of microsporidia on host individuals are frequently subtle and can be context dependent. A key example of the latter comes from a recently discovered microsporidian symbiont of Daphnia, the net impact of which was found to shift from negative to positive based on environmental context. Given this, we hypothesized low baseline virulence of the microsporidian; here, we investigated the impact of infection on hosts in controlled conditions and the absence of other stressors. We also investigated its phylogenetic position, ecology, and host range. The genetic data indicate that the symbiont is Ordospora pajunii, a newly described microsporidian parasite of Daphnia. We show that O. pajunii infection damages the gut, causing infected epithelial cells to lose microvilli and then rupture. The prevalence of this microsporidian could be high (up to 100% in the lab and 77% of adults in the field). Its overall virulence was low in most cases, but some genotypes suffered reduced survival and/or reproduction. Susceptibility and virulence were strongly host-genotype dependent. We found that North American O. pajunii were able to infect multiple Daphnia species, including the European species Daphnia longispina, as well as Ceriodaphnia spp. Given the low, often undetectable virulence of this microsporidian and potentially far-reaching consequences of infections for the host when interacting with other pathogens or food, this Daphnia-O. pajunii symbiosis emerges as a valuable system for studying the mechanisms of context-dependent shifts between mutualism and parasitism, as well as for understanding how symbionts might alter host interactions with resources. IMPORTANCE: The net outcome of symbiosis depends on the costs and benefits to each partner. Those can be context dependent, driving the potential for an interaction to change between parasitism and mutualism. Understanding the baseline fitness impact in an interaction can help us understand those shifts; for an organism that is generally parasitic, it should be easier for it to become a mutualist if its baseline virulence is relatively low. Recently, a microsporidian was found to become beneficial to its Daphnia hosts in certain ecological contexts, but little was known about the symbiont (including its species identity). Here, we identify it as the microsporidium Ordospora pajunii. Despite the parasitic nature of microsporidia, we found O. pajunii to be, at most, mildly virulent; this helps explain why it can shift toward mutualism in certain ecological contexts and helps establish O. pajunii is a valuable model for investigating shifts along the mutualism-parasitism continuum.

First record of a new microsporidium pathogenic to Gonipterus platensis in Brazil.

Microsporidia are naturally occurring fungal-related parasites that can infect nearly all animal hosts, but their biocontrol potential of insect pests is routinely overlooked in agriculture and forestry. This research brings the first report describing the natural occurrence of a microsporidium causing disease in field-collected populations of the invasive eucalyptus snout beetle, Gonipterus platensis (Coleoptera: Curculionidae), a major destructive pest of eucalyptus plantations in Brazil. Adult beetles were collected during field surveys in commercial eucalyptus plantations in southern Brazil to be examined and dissected with typical symptoms to verify presence of microsporidian spores in haemolymph. From 14 plantations in different sites, the natural infection occurrence in these populations ranged from 0 to 65%, while a lab colony exhibited an infection incidence of 70%. Spore density in haemolymph of symptomatic insects averaged 2.1 (± 0.4) × 107 spores/beetle. Symptoms in infected adults were identified by an abnormal abdomen with malformation of the second pair of wings, impairing their flight activity. Electron transmission microscopy of the pathogen showed morphological features similar to species belonging to the genus Nosema or Vairimorpha. Phylogenetic analysis of the full-length small subunit ribosomal RNA gene suggests this pathogen's placement in the genus Vairimorpha, but with a sequence identity of ~ 94% with the nearest neighbours. The low level of sequence identity suggests this pathogen may represent a novel taxon in the genus and further requires whole genome sequencing for definitive taxonomic resolution. These findings provide insights on the natural occurrence of this novel pathogen of this invasive pest in Eucalyptus plantations in Brazil. Further studies are needed to determine potential of this microsporidium in the design of conservative or augmentative biological control programs for this invasive pest.

Tetra disseminated microsporidiosis: a novel disease in ornamental fish caused by Fusasporis stethaprioni n. gen. n. sp.

A novel microsporidial disease was documented in two ornamental fish species, black tetra Gymnocorymbus ternetzi Boulenger 1895 and cardinal tetra Paracheirodon axelrodi Schultz 1956. The non-xenoma-forming microsporidium occurred diffusely in most internal organs and the gill, thus referring to the condition as tetra disseminated microsporidiosis (TDM). The occurrence of TDM in black tetra was associated with chronic mortality in a domestic farmed population, while the case in cardinal tetra occurred in moribund fish while in quarantine at a public aquarium. Histology showed that coelomic visceral organs were frequently necrotic and severely disrupted by extensive infiltrates of macrophages. Infected macrophages were presumed responsible for the dissemination of spores throughout the body. Ultrastructural characteristics of the parasite developmental cycle included uninucleate meronts directly in the host cell cytoplasm. Sporonts were bi-nucleated as a result of karyokinesis and a parasite-produced sporophorous vesicle (SPV) became apparent at this stage. Cytokinesis resulted in two spores forming within each SPV. Spores were uniform in size, measuring about 3.9 ± 0.33 long by 2.0 ± 0.2 µm wide. Ultrastructure demonstrated two spore types, one with 9-12 polar filament coils and a double-layered exospore and a second type with 4-7 polar filament coils and a homogenously electron-dense exospore, with differences perhaps related to parasite transmission mechanisms. The 16S rDNA sequences showed closest identity to the genus Glugea (≈ 92%), though the developmental cycle, specifically being a non-xenoma-forming species and having two spores forming within a SPV, did not fit within the genus. Based on combined phylogenetic and ultrastructural characteristics, a new genus (Fusasporis) is proposed, with F. stethaprioni n. gen. n. sp. as the type species.

Pathogenicity of a microsporidium isolate from the diamondback moth against Noctuid moths: characterization and implications for microbiological pest management.

BACKGROUND: Due to problems with chemical control, there is increasing interest in the use of microsporidia for control of lepidopteran pests. However, there have been few studies to evaluate the susceptibility of exotic species to microsporidia from indigenous Lepidoptera. METHODOLOGY/PRINCIPAL FINDINGS: We investigated some biological characteristics of the microsporidian parasite isolated from wild Plutella xylostella (PX) and evaluated its pathogenicity on the laboratory responses of sympatric invasive and resident noctuid moths. There were significant differences in spore size and morphology between PX and Spodoptera litura (SL) isolates. Spores of PX isolate were ovocylindrical, while those of SL were oval. PX spores were 1.05 times longer than those of SL, which in turn were 1.49 times wider than those of the PX. The timing of infection peaks was much shorter in SL and resulted in earlier larval death. There were no noticeable differences in amplicon size (two DNA fragments were each about 1200 base pairs in length). Phylogenetic analysis revealed that the small subunit (SSU) rRNA gene sequences of the two isolates shared a clade with Nosema/Vairimorpha sequences. The absence of octospores in infected spodopteran tissues suggested that PX and SL spores are closely related to Nosema plutellae and N. bombycis, respectively. Both SL and S. exigua (SE) exhibited susceptibility to the PX isolate infection, but showed different infection patterns. Tissular infection was more diverse in the former and resulted in much greater spore production and larval mortality. Microsporidium-infected larvae pupated among both infected and control larvae, but adult emergence occurred only in the second group. CONCLUSION/SIGNIFICANCE: The PX isolate infection prevented completion of development of most leafworm and beet armyworm larvae. The ability of the microsporidian isolate to severely infect and kill larvae of both native and introduced spodopterans makes it a valuable candidate for biocontrol against lepidopteran pests.

Development, ultrastructural pathology, and taxonomic revision of the Microsporidial genus, Pseudoloma and its type species Pseudoloma neurophilia, in skeletal muscle and nervous tissue of experimentally infected zebrafish Danio rerio.

The microsporidium Pseudoloma neurophilia was initially reported to infect the central nervous system of zebrafish causing lordosis and eventually death. Subsequently, muscle tissue infections were also identified. To understand the infection process, development, and ultrastructural pathology of this microsporidium, larval and adult zebrafish were fed P. neurophilia spores. Spores were detected in the larval fish digestive tract 3-h postexposure (PE). By 4.5-d PE, developing parasite stages were identified in muscle tissue. Wet preparations of larvae collected at 8-d PE showed aggregates of spores in the spinal cord adjacent to the notochord. All parasite stages, including spores, were present in the musculature of larval fish 8-d PE. Adult zebrafish sacrificed 45-d PE had fully developed infections in nerves. Ultrastructural study of the developmental cycle of P. neurophilia revealed that proliferative stages undergo karyokinesis, producing tetranucleate stages that then divide into uninucleate cells. The plasmalemma of proliferative cells has a previously unreported glycocalyx-like coat that interfaces with the host cell cytoplasm. Sporogonic stages form sporophorous vacuoles (SPOV) derived from the plasmalemmal dense surface coat, which "blisters" off sporonts. Uninucleate sporoblasts and spores develop in the SPOV. The developmental cycle is identical in both nerve and muscle. The SPOV surface is relatively thick and is the outermost parasite surface entity; thus, xenomas are not formed. Based on the new information provided by this study, the taxonomic description of the genus Pseudoloma and its type species, P. neurophilia, is modified and its life cycle described.

Ultrastructure and molecular characterization of a microsporidium, Tubulinosema hippodamiae, from the convergent lady beetle, Hippodamia convergens Guérin-Méneville.

Hippodamia convergens, the convergent lady beetle, is available for aphid control in home gardens and in commercial food production systems throughout the United States and Canada. Beetles received from commercial insectaries for biological control are occasionally infected with a microsporidium. The objective of this study was to describe the pathogen by means of ultrastructure, molecular characterization and tissue pathology. All stages of the microsporidium were in direct contact with the host cell cytoplasm. Early developmental stages were proximal to mature spores and both were observed throughout the tissue sections that were examined. Merogony resulted from binary fission. Early-stage sporoblasts were surrounded by a highly convoluted plasma membrane and contained an electron-dense cytoplasm and diplokaryon. Ovoid to elongated late-stage sporoblasts were surrounded by a relatively complete spore wall. The polar filament, polaroplast, and anchoring disk were readily observed within the cell cytoplasm. Mature spores were typical of terrestrial microsporidia, with a thickened endospore surrounded by a thin exospore. Spores contained well-defined internal structures, including a diplokaryon, lamellar polaroplast and a slightly anisofilar polar filament with 10-14 coils arranged in a single or double row. A prominent indentation was evident at the apical end of the spore wall proximal to the anchoring disk. Aberrant spores were also observed. These had a fully developed endospore and exospore but lacked any discernable internal spore structures, and were, instead, filled with lamellar or vesicular structures. Typical and aberrant spores measured 3.58 ± 0.2 × 2.06 ± 0.2 µm (n=10) and 3.38 ± 0.8 × 2.13 ± 0.2 µm (n=10), respectively. Spores were observed in longitudinal muscle surrounding the midgut and within the fat body, Malpighian tubules, pyloric valve epithelium, ventral nerve cord ganglia, muscles and ovaries. The hindgut epithelium was often infected but the connective tissues were rarely invaded. The life cycle and pathology of the microsporidium bears some resemblance to Nosema hippodamiae, the only microsporidium reported from H. convergens by Lipa and Steinhaus in 1959. Molecular characterization of the pathogen genomic DNA revealed that it is 99% similar to Tubulinosema acridophagus and T. ratisbonensis, two pathogens that infect Drosophila melanogaster and 98% similar to T. kingi from D. willistoni. Based on similarities in pathogen ultrastructure and the molecular information gained during this study, we propose that the microsporidium in H. convergens be given the name Tubulinosema hippodamiae.

Occurrence of Nosema oryzaephili in Cryptolestes ferrugineus and transfer to the genus Paranosema.

A microsporidium that closely resembles Paranosema species at the level of the light microscope was isolated from the rusty grain beetle, Cryptolestes ferrugineus. It's identity as Nosema oryzaephili (originally described from Oryzaephilus surinamensis) was confirmed by comparison with a known isolate of N. oryzaephili based on spore size, small subunit rDNA sequence, and relative infectivity to O. surinamensis, Tribolium castaneum, and Ephestia kuehniella. Phylogenetic analysis of the small subunit rDNA indicates clearly that this species belongs in the genus Paranosema and thus the designation Paranosema oryzaephili (Burges, Canning and Hurst) is proposed. In spite of the abundance, economic importance, and world-wide distribution of C. ferrugineus, this is the first report of a microsporidial infection in this species. This is also the first report of P. oryzaephili in the new world.

Effects of a novel microsporidium on the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae).

A newly discovered microsporidium infecting the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae), provisionally placed in the genus Canningia, was studied to determine its impact on O. sulcatus. O. sulcatus populations from several locations were sampled and evaluated for microsporidiosis. A very low prevalence of the disease was observed in all locations surveyed (<3.0%). Laboratory studies were conducted by orally exposing both larvae and adults of O. sulcatus to varying concentrations of Canningia sp. spores. Larval bioassays at a variety of dosages (0, 10, etc.) were performed to evaluate pathogen infectivity, larval survival and growth. Adult bioassays (dosages: 0, 10, etc.) were performed to evaluate longevity, fecundity and mechanisms of vertical pathogen transmission. Larvae and adults were infected in all spore treatments. Larval growth was significantly reduced at dosages above 10 spores/larva. Adults infected at all dosages experienced high levels of mortality and fecundity was reduced to zero. Greenhouse trials were performed to determine if larvae feeding in soil acquired infections when spores were topically applied as a drench application (0, 10(5), 10(6), 10(7) spores/pot). Established larvae feeding on plant roots in pots developed infections when exposed to drench treatments of 10(6) and 10(7) spores/pot after 14-21 days. Canningia sp. is an acute pathogen of O. sulcatus infective to both larvae and adults. Topically applied spores also infected larvae feeding on roots in soilless potting media, suggesting the possibility of using this pathogen in a microbial control program.

Prevalence and impact of the microsporidium Thelohania solenopsae (Microsporidia) on wild populations of red imported fire ants, Solenopsis invicta, in Louisiana.

We surveyed 165 sites to determine the ecological factors that might influence the distribution and prevalence of Thelohania solenopsae, and its effect on the demography of the red imported fire ant (Solenopsis invicta) in Louisiana. The microsporidium was found in 9.9% of colonies and at 16% of sites. Its distribution was clumped within the state with the majority of infected colonies and sites occurring in two infection patches. The proportion of polygyne colonies was a strong (positive) predictor of the proportion of infected colonies at a site. Infected monogyne colonies, however, still accounted for nearly 20% of infected colonies, a much higher proportion than anticipated. Several other factors, including the numbers of colonies at a site, precipitation, proximity to commercial waterways and ports, and type of habitat were also retained in the multiple logistic regression model describing T. solenopsae prevalence. The microsporidium appears to adversely affect the occurrence of brood, and possibly the size of S. invicta colonies and the mass of workers. It, however, was not included in the multiple regression model of the number of colonies or the density of ants at a site. Although our findings do not imply causation, they have identified several variables that might influence the epizootiology of T. solenopsae. Future work should concentrate on experimentally manipulating these variables to confirm these relationships.

Morphological and molecular investigations of a microsporidium infecting the European grape vine moth, Lobesia botrana Den. et Schiff., and its taxonomic determination as Cystosporogenes legeri nov. comb.

We have isolated a microsporidium from a laboratory stock of the European grape vine moth, Lobesia botrana Den. et Schiff. (Lepidoptera, Tortricidae). Screening of this stock showed an infection rate of more than 90%, whereas field collected larvae from three different locations in Rhineland-Palatinate (Germany) did not demonstrate any signs of infection. Light and electron microscopic investigations of infected insects showed that gross pathology, morphology, and ultrastructure of the microsporidium are similar to those described earlier for Pleistophora legeri. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods showed that our isolate was closely related to Cystosporogenes operophterae. Based on our morphological and molecular investigations we propose to rename this species Cystosporogenes legeri nov. comb.

Infection of the cockle Cerastoderma edule in the Baie des Veys (France) by the microsporidian parasite Steinhausia sp.

We report the occurrence of the microsporidian parasite Steinhausia sp. in the oocytes of the common cockle Cerastoderma edule in a natural population in France, where high mortalities occurred. Steinhausia sp. appeared primarily as sporocysts containing many small spores, and putative earlier developmental stages were also observed. Both its prevalence and infection intensity were low, and no host defence reaction was recognized, suggesting that Steinhausia sp. had no detrimental effect on C. edule. Its prevalence was higher in cockles lying on the sediment surface, but the significance of this observation could not be explained given the poor knowledge of the Steinhausia life cycle. The present data did not allow specific identification of the parasite, and further studies are required to determine whether Steinhausia sp. in the cockle is a new species, or a microsporidian infecting multiple host species.

[Study on the biology feature and pathogenicities to silkworm of a microsporidium isolated from Barathra brassicae L].

A microsporidium (called as Bab-M) was isolated from Barathra brassicae L. captured from suburban vegetable plot of Guang Zhou. The spores were long-ovoid in shape and 4.02 +/- 0.36 microns x 1.99 +/- 0.36 microns in size. Immunologically the microsporidium shared spore surface specific antigen(s) with N. bombycis. The ultrastructure and life cycle of Bab-M were similar to that of N. b.. The rate of transovarian transmission was high. The initial conclusion was that Bab-M should be referred to as Nosema bombycis, but there was variation between them.