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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.

Background Microsporidia are obligate intracellular parasites that can infect nearly all invertebrates and vertebrates, posing a threat to public health and causing large economic losses to animal industries such as those of honeybees, silkworms and shrimp. However, the global epidemiology of these pathogens is far from illuminated. Methods Publications on microsporidian infections were obtained from PubMed, Science Direct and Web of Science and filtered according to the Newcastle-Ottawa Quality Assessment Scale. Infection data about pathogens, hosts, geography and sampling dates were manually retrieved from the publications and screened for high quality. Prevalence rates and risk factors for different pathogens and hosts were analyzed by conducting a meta-analysis. The geographic distribution and seasonal prevalence of microsporidian infections were drawn and summarized according to sampling locations and date, respectively. Results Altogether, 287 out of 4129 publications up to 31 January 2020 were obtained and met the requirements, from which 385 epidemiological data records were retrieved and effective. The overall prevalence rates in humans, pigs, dogs, cats, cattle, sheep, nonhuman primates and fowl were 10.2% [2429/30,354; 95% confidence interval (CI) 9.2–11.2%], 39.3% (2709/5105; 95% CI 28.5–50.1%), 8.8% (228/2890; 95% CI 5.1–10.1%), 8.1% (112/1226; 95% CI 5.5–10.8%), 16.6% (2216/12,175; 95% CI 13.5–19.8%), 24.9% (1142/5967; 95% CI 18.6–31.1%), 18.5% (1388/7009; 95% CI 13.1–23.8%) and 7.8% (725/9243; 95% CI 6.4–9.2%), respectively. The higher prevalence in pigs suggests that routine detection of microsporidia in animals should be given more attention, considering their potential roles in zoonotic disease. The highest rate was detected in water, 58.5% (869/1351; 95% CI 41.6–75.5%), indicating that water is an important source of infections. Univariate regression analysis showed that CD4+ T cell counts and the living environment are significant risk factors for humans and nonhuman primates, respectively. Geographically, microsporidia have been widely found in 92 countries, among which Northern Europe and South Africa have the highest prevalence. In terms of seasonality, the most prevalent taxa, Enterocytozoon bieneusi and Encephalitozoon , display different prevalence trends, but no significant difference between seasons was observed. In addition to having a high prevalence, microsporidia are extremely divergent because 728 genotypes have been identified in 7 species. Although less investigated, microsporidia coinfections are more common with human immunodeficiency virus and Cryptosporidium than with other pathogens. Conclusions This study provides the largest-scale meta-analysis to date on microsporidia prevalence in mammals, birds and water worldwide. The results suggest that microsporidia are highly divergent, widespread and prevalent in some animals and water and should be further investigated to better understand their epidemic features. Graphical Abstract

Background Microsporidia MB , a Plasmodium -transmission-impairing symbiont in Anopheles arabiensis , has malaria control potential. This study assessed its prevalence and phylogeny in An. gambiae s.l. and An. funestus in Busia, Kenya and investigated the influence of environmental factors on its occurrence. Additionally, the prevalence of key insecticide resistance mutations in these mosquito populations was determined. Methods Mosquito larvae and adults were collected from three sub-counties in Busia County, Kenya and identified based on morphological characteristics. PCR was used to determine Anopheles species distribution and Microsporidia MB prevalence following DNA extraction from the samples. Insecticide resistance target-site mutations were identified using TaqMan genotyping in a subset of the mosquito samples. Multivariable logistic regression models were used to assess associations of Microsporidia MB infection and ecological factors. Microsporidia MB -positive samples were whole-genome sequenced and phylogenetically analysed. Results Overall, An. gambiae s.l. (including An. gambiae s.s. and possibly An. coluzzii ) comprised 57.3% of samples analysed while An. funestus comprised 25.7% and An. arabiensis 17% and their distribution varied significantly across the three sub-counties (Chi-square, χ² = 577.44, df = 4, p  < 0.001). Microsporidia MB prevalence was low to moderate (0 to 6.4%) and highest in An. gambiae s.l. Anopheles gambiae s.l. showed significantly higher odds of infection compared to An. arabiensis (aOR = 5.94, 95% CI: 1.96–26.77, p  = 0.006). Larvae reared to adults had significantly lower odds of infection than indoor-collected adults (aOR = 0.48, 95% CI: 0.26–0.86, p  = 0.014). Insecticide resistance genotyping revealed high frequencies of kdr- East (94.7%) and kdr- West (60%) mutations in the Anopheles subset analysed, while Ace-1 and GSTe2 mutations were absent. Phylogenetic analysis placed Microsporidia MB isolated from Busia, within Clade IV, closely related to the originally sequenced Ahero reference, but still distinct from other microsporidian clades (I and III). Conclusion The present study highlights the occurrence of Microsporidia MB in multiple Anopheles vectors associated with malaria suggesting its broader potential as a vector control tool. The high prevalence of kdr mutations indicate a significant challenge to insecticide-based vector control in the region. Further investigation into the phenotypic expression of insecticide resistance in these populations is important. Results of the phylogenetic analysis suggest a common ancestry for Microsporidia MB isolates from Busia with the Ahero reference one, highlighting shared traits with potential for malaria control.

Background Microsporidia MB disrupts Plasmodium development in Anopheles mosquitoes, making it a possible biocontrol tool for malaria. As a tool for vector/disease control, its ecological distribution and the factors that determine their occurrence must be defined. We investigated the frequency of Microsporidia MB in Anopheles mosquitoes across selected sites in northern and southern Ghana, as well as the physicochemical parameters of mosquito breeding water that are associated with the occurrence of the fungus, by fitting regression models. Methods A non-column extraction method was used to extract DNA from the abdomens of 4255 adult Anopheles mosquitoes that emerged from larvae and pupae collected between August and October of 2021 and 2022. Detection of Microsporidia MB was achieved using quantitative PCR (qPCR), while mosquito species were molecularly identified using short interspersed nuclear elements (SINE), restriction fragment length polymorphism (RFLP) methods, and the ANOSPP algorithm. Results Overall Microsporidia MB distribution was 2.2% (92/4255). Male mosquitoes exhibited a higher frequency of infections and had a predicted probability of infection that was 85% higher than that of females. Sites in Ghana's Savannah zone had the highest Microsporidia MB distribution (68.5%). Biochemical oxygen demand in mosquito breeding water was estimated to be positively associated with and significantly predicts Microsporidia MB in mosquitoes with an accuracy of 94%. Increasing ammonium ion concentrations reduced the chances of finding Microsporidia MB -positive mosquitoes. According to our data, all Anopheles mosquitoes, including minor species such as An. squamosus , An. pretoriensis and An. rufipes , had equal probability of Microsporidia MB infection. Conclusions These results provide preliminary information on micro-ecological factors that potentially support the sustainability of Microsporidia MB infection in mosquitoes during their aquatic life stages. It will be important, therefore, to explore the impact of strategies for larval source management on these factors to ensure that the symbiont's persistence during the host's aquatic stages may not be adversely affected should it be used as an integrated approach for mosquito/disease control. Graphical abstract

Recent discoveries of Microsporidia MB in Anopheles mosquitoes and its demonstrated capacity to block malaria transmission suggest that it could be a promising candidate for a symbiont-based malaria control strategy. However, the diversity of Microsporidia MB , particularly in major malaria vector such as An. coluzzii , remains unstudied in Cameroon. In this study, we investigated the prevalence and phylogenetic analysis of Microsporidia MB , along with its maternal transmission in natural populations of Anopheles mosquitoes in Cameroon. An entomological survey was conducted across four localities in Cameroon. PCR targeting the ribosomal RNA gene was used to detect the Microsporidia MB 18S sequence. Positive samples were validated by sequencing and phylogenetic analysis. Subsequently, the maternal transmission of Microsporidia MB was assessed by detecting its presence in F 1 mosquitoes derived from F 0 females that tested positive. Additionally, we examined the potential correlation between Microsporidia MB and P. falciparum . A total of 2559 mosquitoes, belonging to three species ( An. coluzzii , An. arabiensis , and An. gambiae ) were collected. Microsporidia MB infection was detected exclusively in An. coluzzii from Gounougou, with a prevalence of 1.13% (n = 29). Phylogenetic analysis showed that the Microsporidia MB strains detected in Cameroon were closely related to those found in Kenya. Evidence of vertical transmission was confirmed, with rates ranging from 50 to 100%, from F 0 parents to F 1 offspring, regardless of sex. Notably, none of the Microsporidia MB -positive mosquitoes carried P. falciparum . This study provides field-based evidence of Microsporidia MB presence in Anopheles mosquitoes in Cameroon. Further research should explore the stability of vertical transmission and the influence of this Microsporidia MB symbiont on Plasmodium development.

Introduction The Plasmodium transmission-blocking endosymbiont Microsporidia MB was previously identified in Anopheles gambiae s.l., but its association with the carriage of the genotypes of the L1014F kdr mutation, as well as the ecological factors driving its geographical distribution remain understudied. Methods Adult mosquitoes were field-collected using human landing catches (HLCs) across 60 villages in the Covè, Ouinhi, and Zangnanado communes of southern Benin. After morphological identification, a sub-sample of An. gambiae s.l. were molecularly speciated, and genotypied for the L1014F kdr mutation by Polymerase Chain Reaction (PCR). Enzyme-Linked Immunosorbent Assay (ELISA) and qPCR were also used to assess infection with Plasmodium falciparum sporozoites and Microsporidia MB, respectively. The environmental variables that drive the habitat suitability for Microsporidia MB were also assessed using Maximum Entropy (MaxEnt) modelling. Results The An. gambiae complex ( N  = 1040) was composed of 93.7% An. coluzzii , 4.4% An. gambiae s.s., 0.2% An. gambiae s.s./ coluzzii hybrids, while the rest failed to amplify. Infection prevalence with Microsporidia MB was 1.6% (95% CI: 0.7–3.3) in An. coluzzii and 2.2% (95% CI: 0.1–13.2) in An. gambiae s.s. The P. falciparum sporozoite rate was 2% (95% CI: 1.2–3.1, N  = 974) in An. coluzzii , and null in An. gambiae s.s. ( N  = 46). None of the mosquitoes infected with Microsporidia MB were infected with P. falciparum . The frequency of the L1014F kdr mutation was 75.1% (95% CI: 73.1–76.9) in An. coluzzii and 91.3% (95% CI: 83.1–95.9) in An. gambiae s.s. Microsporidia MB was absent in kdr -SS mosquitoes but was present in low proportions in both kdr -RS and kdr -RR mosquitoes (1.9%, 95% CI: 0.6–5.1). The mean load of Microsporidia MB DNA was higher in kdr -RR (23.23 ng/µl, 95% CI: 18.77–28.48) compared to kdr -RS (10.13 ng/µl, 95% CI: 7.36–13.91) mosquitoes. The elevation and soil contributed to explain, at 78% and 20% respectively, the habitat suitability for Microsporidia MB. Conclusion In this study, we demonstrated that An. gambiae s.l. mosquitoes bearing the L1014F kdr resistant allele was associated with a higher prevalence and load of Microsporidia MB than their susceptible counterparts. Moreover, the geographical distribution of Microsporidia MB was found to be influenced by certain environmental conditions, which warrant further large-scale investigations.

Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 μm⋅s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.

Background Microsporidia MB , a naturally occurring Anopheles symbiont was shown to strongly impair Plasmodium transmission without imposing deleterious fitness effects on larval development, fecundity, adult survival, and adult sex ratio, and for these reasons it is being proposed as a promising tool for malaria control. However, there is a limited knowledge about its ecology, transmission dynamics in the environments with varying abiotic conditions, and whether these could impact on the mode of host transmission. This study aimed to determine the presence and prevalence of Microsporidia MB in rice fields in Benin during both the dry and wet seasons. Methods Anopheles larvae and adults were collected from rice fields and houses around rice fields in two locations (Koussin-Lélé and Magoumi). The collections took place during both the dry and wet seasons. The larvae and adults were molecularly identified to species level using molecular techniques and they were also screened for the presence of Microsporidia MB using PCR following standard protocols. Moreover, breeding sites were also analysed. Results The species identification results revealed that Anopheles coluzzii was the main species in Koussin-Lélé, accounting for 100% of the 1718 samples, while Anopheles gambiae sensu stricto ( s.s .) was predominant in Magoumi accounting for 98.17% of the 986 samples. In Koussin-Lélé, Microsporidia MB prevalence of 14% (n = 276) was observed, whereas in Magoumi, only two samples (0.3%) were tested positive out of 667 screened in the dry season. During the wet season, the prevalence of Microsporidia MB symbiont was low with rates of 0.7% (5) in Koussin-Lélé and 0.6% (8) in Magoumi. However, the prevalence was relatively moderate in adult field collected mosquitoes with 3.09% (n = 1554) followed by larvae samples with 2.93% (n = 682) and adults emerged from field-collected larvae with 2.67% (n = 1235). Analysis of breeding sites revealed high concentrations of nitrate, nitrite and copper in Magoumi during the dry season, which was associated with a low prevalence of Microsporidia MB symbiont in this area. In the wet season, high concentrations of lead and nitrate were recorded in Koussin-Lélé with high concentration of copper in Magoumi, and both localities showed low prevalence of Microsporidia MB infection. Conclusion This study revealed a high prevalence of Microsporidia MB symbiont in Benin during the dry season. Further investigations might be necessary, and modelling of the prevalence and characteristics of breeding sites could help predict the presence of this symbiont in other locations and countries.

Microsporidia sp. MB, a microsporidian symbiont found naturally in mosquitoes, has potential as a novel malaria control tool since it can inhibit development and transmission. The most feasible MB-based control strategy would involve dissemination through live mosquito releases, or release of spores infective to mosquito larvae. To implement either strategy, establishment of stable mosquito colonies carrying MB at a high frequency is likely to be essential. The progeny of field caught from Burkina Faso were isolated for individual egg laying and tested for MB. The progeny of the MB positive females were pooled and this process was repeated for multiple generations. The relative density of MB in different life stages and tissues of the host was examined using a novel duplex qPCR assay. We also examined the impact of MB on egg production through individualization for egg laying and counting of eggs. Finally, we examined laid eggs for presence of MB spores. Three colonies and one hybrid colony were established with high prevalence and density of MB and were maintained for more than two years with minimal intervention. MB prevalence and density was highest in eggs and adult females and lowest in L4 larvae; in adults density was highest in the gonads. Additionally, MB density increased in ovary samples following blood feeding which was likely due to the activation of sporogony. The production of spores is the reason why MB-carrying females lay more white non-hatching eggs and show a small reduction in the median number of eggs laid. Establishment of several stable MB carrying colonies and understanding the impact of spores on egg production are significant steps forward in developing MB as a malaria control tool.

A new microsporidian species, Globosporidium paramecii gen. nov., sp. nov., from Paramecium primaurelia is described on the basis of morphology, fine structure, and SSU rRNA gene sequence. This is the first case of microsporidiosis in Paramecium reported so far. All observed stages of the life cycle are monokaryotic. The parasites develop in the cytoplasm, at least some part of the population in endoplasmic reticulum and its derivates. Meronts divide by binary fission. Sporogonial plasmodium divides by rosette-like budding. Early sporoblasts demonstrate a well-developed exospore forming blister-like structures. Spores with distinctive spherical shape are dimorphic in size (3.7 ± 0.2 and 1.9 ± 0.2 μm). Both types of spores are characterized by a thin endospore, a short isofilar polar tube making one incomplete coil, a bipartite polaroplast, and a large posterior vacuole. Experimental infection was successful for 5 of 10 tested strains of the Paramecium aurelia species complex. All susceptible strains belong to closely related P. primaurelia and P. pentaurelia species. Phylogenetic analysis placed the new species in the Clade 4 of Microsporidia and revealed its close relationship to Euplotespora binucleata (a microsporidium from the ciliate Euplotes woodruffi), to Helmichia lacustris and Mrazekia macrocyclopis, microsporidia from aquatic invertebrates.