Microsporidian Infection Research Articles

Molecular detection and phylogenetic analysis of microsporidia in stool specimens isolated from multiple sclerosis patients in the west of Iran.

Intestinal microsporidiosis is an emerging opportunistic infection that primarily affects individuals with compromised immune systems. This study investigated intestinal microsporidia infections in individuals diagnosed with multiple sclerosis (MS) and elucidated the genetic diversity and evolutionary relationships of microsporidia. A total of 116 stool samples were collected from individuals diagnosed with MS, including 54 men and 63 women, during 2022-2023 in Kurdistan Province, western Iran. The mean age of the participants was 38.28±7.8years. RNA extraction was performed, followed by amplification of the small subunit ribosomal RNA (SSU rRNA) to identify and characterize microsporidia and their associated genetic markers. A phylogenetic tree was constructed using MEGA X software to explore the evolutionary relationships among the isolates. PCR revealed Enterocytozoon bieneusi in 5 of 116 samples (4.3%), whereas no positive cases of Encephalitozoon species were detected. Additionally, no statistically significant associations were observed between the presence of microsporidia and variables such as age, sex, or geographic region. This study highlights the clinical significance of Enterocytozoon bieneusi in immunocompromised populations, particularly in patients with multiple sclerosis (MS). The findings underscore the potential role of zoonotic transmission and highlight the urgent need for enhanced diagnostic capabilities and preventive strategies to combat intestinal microsporidiosis in vulnerable groups, particularly in regions such as western Iran.

First Record and Identification of a Microsporidian Pathogen, Nosema Maddoxi in the Population of Brown Marmorated Stink Bug Halyomorpha Halys (Hemiptera: Pentatomidae) in Türkiye

PurposeThe brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), is an invasive and a highly polyphagous species with a strong dispersal capacity. Unfortunately, there is currently no effective control method that can prevent or reduce the economic loss caused by this pest. Among natural enemies, microsporidia cause infections in insects so that they can generally shorten life span, reduce fertility and inhibit growth.MethodsCurrent study involved field study, light and electron microscopy and molecular phylogenic analyses of a microsporidian pathogen in the populations of H. halys in Türkiye.ResultsThe microsporidian infection were mostly observed in malpighian tubules and fatty tissue filled with high spore density. Fresh uninucleate spores are oval, measued as 3.73 × 2.01 µm in dimensions. The mature spore wall is relatively thick and measures 75–85 nm and consists of a clear endospore (40–50 nm) and an electron-dense, uniform, thin exospore (25–30 nm). The polar filament is isofilar, 85–110 mm in diameter and has 7–8 coils. It is found to be most closely related to Nosema maddoxi isolate Mn.6 isolated from H. halys in Georgia in phylogenetic tree.ConclusionThe spore morphology and structural features of the isolate from H. halys identify it as Nosema maddoxi. The phylogenetic analyses confirm both light and electron microscopic observations. This is the first microsporidian record from H. halys and also from the order Hemiptera in Türkiye. It is also confirmed that the invasive pest, H. halys carries its natural pathogen, N. maddoxi to new geographical locations during its distribution.

Analysis of Possible Coexistence of Microsporidia, Plasmodium falciparum and Wuchereria bancrofti in Anopheles gambiae s.l within Ahmadu Bello University, Zaria, Nigeria.

Anopheles gambiae is a vector of Plasmodium falciparum and Wuchereria bancrofti. Endosymbionts are reported to block development of various parasites in mosquitoes. Microsporidia was reported to affect the development of P. falciparum in mosquitoes. Data on such observation is limited in Nigeria. Therefore, the prevalence of Microsporidia and its coinfection with W. bancrofti and P. falciparum in An. gambiae s.l was studied within Ahmadu Bello University, Zaria. Of the 912 mosquitoes sampled, 124 were An. gambiae s.l The midgut assessment of the Anopheles mosquitoes using light microscopy and polymerase chain reaction (PCR) showed a 12% prevalence of a mono microsporidia infection with no coinfection with either P. falciparum or W. bancrofti. Only 4.03% of the An. gambiae s.l. were found to be coinfected with P. falciparum and W. bancrofti while no mosquito harboured all the microorganisms CONCLUSION: This data further supports the potential of Microsporidia as an antagonist for the development of pathogens in mosquitoes.

Microsporidiosis in Patients with Autoimmune Diseases Undergoing Monoclonal Antibody Associated Therapy.

We present Enterocytozoon bieneusi infection in four patients with autoimmune diseases undergoing prolonged monoclonal antibody therapies. Two patients suffered from inflammatory bowel disease and received anti-TNF therapies, whereas two other patients suffered from systemic lupus erythematosus with renal involvement and received anti-CD20 or anti-BLyS protein therapies. Three out of four patients consulted for diarrhea with abdominal pain without intestinal inflammation or bleeding at the time of sampling. The fourth patient did not declare intestinal troubles. Microsporidia genotype detected in this study were S9, C, Wildboard3 with one patient harboring 2 genotypes S6 and EBCMAP-038. Management of microsporidia infection included albendazole and reduction of immunosuppression treatment, but no specific treatment was implemented in two other patients. In conclusion, microsporidia infection occurs in patients with autoimmune diseases undergoing prolonged monoclonal antibody therapies. Diagnosis should be carefully assessed in this population and a thorough benefit-risk analysis is essential prior to initiating therapeutic interventions.

Prevalence and Individualized Risk Factors of E. bieneusi and E. intestinalis Infections Among People Living with HIV (PLHIV) with Diarrhea in Ecuador: Insights from a Single-Center Cross-Sectional Study.

Background: Microsporidia, particularly E. bieneusi and E. intestinalis, are emerging opportunistic pathogens that pose significant health risks to immunocompromised individuals, especially people living with HIV (PLHIV). Despite the global recognition of microsporidia's impact, there has been limited research on their prevalence and associated risk factors in Ecuador. This study aimed to investigate the prevalence and identify risk factors associated with microsporidia infections among PLHIV with diarrhea in Ecuador. Methods: A cross-sectional study was conducted at the José Daniel Rodríguez Infectious Hospital in Guayaquil, Ecuador, between April 2021 and May 2022. A total of 85 PLHIV with diarrhea were included in the analysis. Fecal samples were tested for microsporidia using fast-hot Gram chromotrope staining, immunofluorescence microscopy, and transmission electron microscopy. Logistic regression was performed to assess the association between demographic, clinical, and behavioral factors and microsporidia infection. Results: The prevalence of microsporidia infections among the study population was 18.8%. Logistic regression analysis identified significant associations with age ≥ 60 years (OR = 19.169, p = 0.036), female sex (OR = 10.491, p = 0.028), and non-adherence to antiretroviral therapy (OR = 8.466, p = 0.077). Marginally significant associations were observed for substance use (OR = 0.262, p = 0.094) and high/very high HIV viral load (≥10,000 copies/mL, OR = 0.457, p = 0.094). CD4 count and marital status showed descriptive trends but were not statistically significant. Conclusions: This study highlights the burden of microsporidia infections among PLHIV in Ecuador and underscores the need for targeted public health interventions. Strategies should prioritize older individuals, females, and those with poor ART adherence to reduce infection risk. Future research is needed to explore additional risk factors and refine precision medicine approaches for this population.

CLINICAL PROFILE OF MICROSPORIDIAL KERATITIS AT A TERTIARY EYE CARE CENTER IN BUNDELKHAND REGION

Background: Although rare, microsporidia are sometimes misdiagnosed as the cause of corneal infections. The ubiquitous, obligatory, intracellular parasite is the causal agent. In Asia, the prevalence of microsporidiosis is rising in tandem with the monsoon season. Immunosuppression (HIV infection, topical corticosteroids), contact lens usage, and injuries from water or dirt exposure are risk factors. Purpose: To study clinical profile of patient with microsporidial keratitis in department of ophthalmology at M.L.B. Medical College Jhansi between April to October 2024. To summarizes the current literature on the etiology and clinical presentation of microsporidial infections of the cornea and highlights ongoing developments in available diagnostic modalities and treatment regimens. Materials and Methods: Prospective study of 20 patients of microsporidial keratitis present in Department of Ophthalmology at M.L B Medical College Jhansi. Results: A total of 20 patients were examined out of which 19 patients (95%) presented with pain and redness, 18 patients (90%) present with foreign body sensation and blurring of vision, 15 patients (75%) present with photophobia, 14 patients (70%) present with lid swelling. 18 patients (90%) have conjunctival hyperemia, 16 patients (50%) have diffuse punctate epitheliopathy and 1 have deep stromal infiltrateson examination. Conclusion: Most commonly patients presents with redness and eye pain, followed by foreign body sensation, blurring of vision, photophobia and least commonly with lid swelling.

MiR-PC-3p-241582_34 Contributes to the Infection of Athetis lepigone by Regulating the Expression of HSWP4 in Nosema bombycis.

Athetis lepigone is a recurring pest in the maize seedling stage under the wheat-maize no-tillage direct seeding system in China's summer maize region. Our previous research identified a highly pathogenic Nosema bombycis to A. lepigone, which spore wall protein plays an important role in the infection process. However, the regulatory mechanism of this spore wall protein is still unclear. In this study, we explored the regulatory mechanism of miRNAs on spore wall proteins. Transcriptome sequencing results showed that expression of the spore wall protein, HSWP4, significantly increased in the germination group compared to dormancy group. Silencing of HSWP4 reduced the number of microsporidian spores breaking through the midgut wall cells of A. lepigone. Association analysis of small RNA and mRNA revealed that the targeting site of miR-PC-3p-241582_34 on HSWP4 was located in the CDS region, and miR-PC-3p-241582_34 had a significant negative regulatory relationship with HSWP4. The dual luciferase reporter assay demonstrated that miR-PC-3p-241582_34 significantly affected the luciferase activity of the HSWP4-3'UTR expression vector (P < 0.05). Delivery of miRNA mimics decreased the expression of HSWP4 and inhibited the behavior of microsporidian spores breaking through the midgut wall of A. lepigone. On the other hand, delivery of inhibitors produced opposite results, indicating that the miR-HSWP4 pathway plays an important role in microsporidian infection of A. lepigone. This study provides a new theoretical basis for understanding the pathogenic mechanism and gene regulation of microsporidia, as well as for the green control of A. lepigone.

Distribution and evidence of co-infection of the two microsporidian parasites Astathelohania contejeani and Nosema austropotamobii in Austropotamobius pallipes complex in Northern and Central Italy.

Austropotamobius pallipes complex is an endangered freshwater crayfish species in Europe and the assessment of the health status of its wild populations is essential for conservation purposes. The two microsporidia Astathelohania contejeani and Nosema austropotamobii have been reported to cause in A. pallipes complex a chronic parasitic infection, known as ‘porcelain disease’, which reduces population fitness and leads the host to death. Due to the similar macroscopic signs produced, molecular biology analyses are required to unambiguously distinguish between these microsporidia. Focusing on A. pallipes complex populations located in Northern and Central Italy, the present study provides an evaluation of prevalence and distribution of A. contejeani and N. austropotamobii, and investigates the variables affecting the probability of detecting infected specimens during a survey (e.g. sex, crayfish density, longitude). Microsporidia were identified in 12 populations among the 42 monitored from 2011 to 2017, with an average prevalence of 3.12% for A. contejeani and 3.60% for N. austropotamobii, the latter being reported in a wider area than previously documented (from Lombardy to Liguria Regions). Notably, crayfish co-infected by both microsporidia were also detected in 4 populations. Moreover, it was observed that the probability of detecting a crayfish with a microsporidian infection significantly increased eastwards in the studied area, especially for N. austropotamobii. Our distribution map for microsporidiosis, combined with molecular screening, will be useful for planning breeding and translocation efforts for A. pallipes complex populations.

Extensive influence of microsporidian infection on sucrose solution consumption, antioxidant enzyme activity, cell structure, and lifespan of Asian honeybees.

Apis cerana is the original host of Vairimorpha (Nosema) ceranae, a widespread fungal parasite that causes bee nosemosis, which severely threatens the health of bee colonies and the sustainable development of the apiculture industry. To evaluate the impact of V. ceranae infection on A. c. cerana workers, V. ceranae spores were purified and used to inoculate newly emerged workers to evaluate the effects of V. ceranae infection. This was followed by an in-depth investigation of V. ceranae spore load and host sucrose solution consumption. Activities of four major antioxidant enzymes (SOD, PPO, CAT, and GST) were determined. Paraffin sections of the host midgut tissue were prepared and subjected to microscopic observation. The survival rates of V. ceranae-inoculated and uninoculated workers were analyzed. The results showed that spore load gradually increased and peaked at 12 dpi. The consumption of workers in the V. ceranae-inoculated group was extremely significant higher (P < 0.0001) than that of workers in the un-inoculated group. The results of antioxidant enzyme activity were suggestive of positive host defense via catalase (CAT) and glutathione-S-transferase (GST) in the middle stage of infection, as well as the negative fungal impact on superoxide dismutase (SOD) and polyphenol oxidase (PPO) at the whole stage of infection, reflecting the complex host-parasite interaction. Additionally, we observed a disruption in the structure of the host midgut epithelial cells. Moreover, the survival rate of workers in V. ceranae-inoculated groups was nearly always lower than that of workers in the uninoculated groups. These results demonstrate a consistent increase in spore load with the proliferation of V. ceranae, leading to persistent energetic stress and midgut epithelial cell structural damage to the host, ultimately resulting in a shortened lifespan for the host. Our findings enhance the current understanding of the interactions between A. cerana and V. ceranae as well as provide a solid basis for exploring the mechanisms underlying host response and V. ceranae infection.

Molecular Survey of Microsporidia, Blastocystis, Cryptosporidium and Giardia in Pet Avian Species in Tehran, Iran

Background: Opportunistic microorganisms of the intestinal tract, such as Cryptosporidium spp. Giardia spp. Blastocystis sp. and microsporidia, are increasingly responsible for clinical disorders in various host species, including humans. Objectives: This study was conducted to search for the above parasites in the feces of pet birds using parasitological and molecular methods in Tehran City, Iran. Methods: In the current study, fecal samples of avian birds were collected and investigated with modified Ziehl-Neelsen, modified trichrome, and trichrome staining for the presence of microsporidia, Cryptosporidium, Blastocystis and Giardia. All the samples were examined molecularly with specific primers and PCR methods. Results: Three of the examined droppings contained Encephalitozoon hellem genotype 1B (2%) by PCR and sequencing. The microsporidian organisms were recovered from the droppings of a clinically normal green-cheek parakeet, an African gray parrot, and a lovebird. Other parasites that were examined were not found in the analyzed samples. Conclusion: The current study proved that captive pet birds are a possible source of microsporidian infection. Besides the fact that encephalitozoonosis is predominantly subclinical in birds, the highly resistant nature of the microsporidia spores can put the owners at increased risk of disease acquisition via spore inhalation or ingestion.

Microsporidian EnP1 alters host cell H2B monoubiquitination and prevents ferroptosis facilitating microsporidia survival

Microsporidia are intracellular eukaryotic pathogens that pose a substantial threat to immunocompromised hosts. The way these pathogens manipulate host cells during infection remains poorly understood. Using a proximity biotinylation strategy we established that microsporidian EnP1 is a nucleus-targeted effector that modifies the host cell environment. EnP1's translocation to the host nucleus is meditated by nuclear localization signals (NLSs). In the nucleus, EnP1 interacts with host histone H2B. This interaction disrupts H2B monoubiquitination (H2Bub), subsequently impacting p53 expression. Crucially, this inhibition of p53 weakens its control over the downstream target gene SLC7A11, enhancing the host cell's resilience against ferroptosis during microsporidian infection. This favorable condition promotes the proliferation of microsporidia within the host cell. These findings shed light on the molecular mechanisms by which microsporidia modify their host cells to facilitate their survival.

Translocator protein (TSPO) inhibits Nosema bombycis proliferation in silkworm, Bombyx mori.

Pebrine disease, caused by Nosema bombycis (Nb) infection in silkworms, is a severe and long-standing disease that threatens sericulture. As parasitic pathogens, a complex relationship exists between microsporidia and their hosts at the mitochondrial level. Previous studies have found that the translocator protein (TSPO) is involved in various biological functions, such as membrane potential regulation, mitochondrial autophagy, immune responses, calcium ion channel regulation, and cell apoptosis. In the present study, we found that TSPO expression in silkworms (BmTSPO) was upregulated following Nb infection, leading to an increase in cytoplasmic calcium, adenosine triphosphate, and reactive oxygen species levels. Knockdown and overexpression of BmTSPO resulted in the promotion and inhibition of Nb proliferation, respectively. We also demonstrated that the overexpression of BmTSPO promotes host cell apoptosis and significantly increases the expression of genes involved in the immune deficiency and Janus kinase-signal transducer and the activator of the transcription pathways. These findings suggest that BmTSPO activates the innate immune signalling pathway in silkworms to regulate Nb proliferation. Targeting TSPO represents a promising approach for the development of new treatments for microsporidian infections.

Tick-borne microsporidiosis: ticks as a neglected source of human microsporidian infections?

ABSTRACT We detected 24 Encephalitozoon cuniculi positive Ixodes ricinus ticks of 284 collected in the Czech Republic. Since the route of transmission of microsporidia is not fully understood, the presence of microsporidia in ticks raises the question of whether they may be involved in the transmission of these pathogens.

Mass mortality event of round sardinella Sardinella aurita Valenciennes associated with Glugea Thélohan, 1891 microsporidian infection off the southern Italian coast.

Intracellular parasites of the genus Glugea Thélohan, 1891 (Microsporidia) comprise about 34 putative species capable of causing high morbidity and mortality in freshwater and marine teleost fishes. In this study, we report on the first mass mortality event associated with Glugea sp. infecting free-ranging round sardinella Sardinella aurita in the southern Tyrrhenian Sea (Italy). Here, we describe the ultrastructure of mature spores of this microsporidian and characterize it molecularly, as well as report its phylogenetic position. Most of the affected fish showed an irregular swelling of its abdomen. At necropsy, a variable number of xenomas, spherical to ellipsoidal in shape, were found in the peritoneal cavity strongly attached to the viscera of all fish. Histological analysis revealed varying severity of chronic inflammation along with occasional necrosis in visceral organs associated with multiple xenoma proliferation. These pathological findings were considered the main cause of this mass mortality event. Morphologically, the present material was closely related to G. sardinellesis and G. thunni. The phylogenetically closest taxa to the newly SSU rDNA sequence were G. thunni and an erroneusly identified G. plecoglossi, which were very closely related to each other, also suggesting that all these sequences might belong to the same species.

Detection of Enterocytozoon bieneusi in Non-Human Primates in Portuguese Zoos.

Enterocytozoon bieneusi, an intracellular eukaryote closely related to fungi, is recognized as a significant pathogen affecting humans, particularly those with compromised immune systems. While its transmission routes are still not fully elucidated, fecal-oral transmission remains the primary one. With a wide host range, the zoonotic potential of E. bieneusi is a concern, albeit direct evidence of animal-to-human transmission remains scarce. Genotyping based on the internal transcribed spacer (ITS) region facilitates the delineation of genetic diversity, with potentially zoonotic genotypes predominantly associated with Groups 1 and 2. Despite the broad spectrum of susceptible animal hosts, research into microsporidian infection among zoo animals remains limited. This study aimed to evaluate the occurrence of E. bieneusi infection across diverse captive animals, focusing on zoo settings in Portugal. Fecal samples were collected from a variety of animals, and molecular detection of E. bieneusi was conducted using nested PCR targeting the ITS region. Of 127 fecal samples, 1.57% (95% CI: 0.19-5.57) tested positive for E. bieneusi, with non-human primates (NHP's) exhibiting an 18.18% (95% CI: 2.28-51.78) occurrence. Phylogenetic analysis revealed clustering within Group 2 genotypes, indicating potential zoonotic implications. This study highlights the need for further research to understand the epidemiology of E. bieneusi in zoo environments and its potential transmission pathways to humans.

Endochitinase from the Microsporidia Nosema ceranae facilitates infection in the honey bee Apis mellifera

Bees are crucial pollinators in terrestrial ecosystems, responsible for 80% of insect-driven pollination and playing a vital role in the pollination of 75% of crops. The honey bee, Apis mellifera, is not only used in honey production but also serves as a pollinator in agriculture. However, A. mellifera faces various challenges, including exposure to pathogens such as the Microsporidia Nosema ceranae, which has been linked to decreased crop yields and colony losses. Nosema ceranae spores infect adult honey bees by penetrating the midgut lumen and invading the cytoplasm of epithelial cells, completing their life cycle. However, the midgut possesses a protective mechanical barrier called the peritrophic matrix, composed of chitin and proteins, which prevents epithelial infection. Nevertheless, N. ceranae overcomes this primary defense mechanism, though the specific mechanisms it employs to cross the peritrophic matrix and reach the midgut epithelium are not yet well understood. This study aimed to investigate the potential role of the predicted endochitinase from N. ceranae to infect bees. We tested the hypothesis that inhibiting the expression of N. ceranae endochitinase through RNA interference would impact the pathogen infection of A. mellifera. Bees treated with dsRNA targeting endochitinase, administered 12 and 24 h after spore inoculation, exhibited suppressed endochitinase gene expression and a decrease in the number of total and viable N. ceranae spores in the midgut. These results indicate that inhibiting the expression of the target gene through RNA interference affects Microsporidia infection, underscoring the importance of this enzyme in the infection process.

Endoparasite Infections in Captive Inland Bearded Dragons (Pogona vitticeps) in Italy.

The inland bearded dragon (Pogona vitticeps) is a lizard species commonly kept as a pet worldwide. Endoparasites are among the most important pathogens affecting bearded dragons. The aim of this study was to evaluate the endoparasites in captive P. vitticeps in Italy. Faecal samples from 30 P. vitticeps were analysed by fresh faecal smears, flotation tests, the Mini-FLOTAC technique, and a rapid immunoassay to detect Cryptosporidium spp. To search for microsporidia, PCR and sequencing were performed on the faecal samples. Data were statistically analysed. The overall positivity rate for endoparasites was 83.3% (25/30). The identified endoparasites were oxyurids (17/30, 56.7%), Isosospora amphiboluri (13/30, 43.3%), Encephalitozoon pogonae (4/18, 22.22%), and Cryptosporidium sp. (1/30, 3.33%). The positivity for protozoa was significantly higher in juveniles compared to adults. Moreover, the frequency of clinical signs was significantly higher in the positive animals. The results obtained here emphasize the importance of regular veterinary examinations of captive P. vitticeps, aimed at the diagnosis, treatment, and control of endoparasites. This study is one of the largest surveys on microsporidia infections in living bearded dragons, suggesting that E. pogonae may be widespread in this lizard.

Microsporidian heavy infection in a batch of salted and dried cod.

The aim of this work is the description and characterization of a severe microsporidian infection in a batch of salted and dried cod. Particularly, the case involves a batch of approximately 800 kg obtained from Gadus macrocephalus (Food and Agriculture Organization Zone 61 - Northwest Pacific Ocean), which, after rehydration and sectioning operations, underwent routine company checks before packaging. In about 20% of the samples, the presence of whitish nodules with a diameter ranging from 1 to 2 mm was observed on the surface of the fillets and in cross-section. The lesions ranged from a few units to 10 per cm2. Some samples were subjected to fresh microscopic observation with the stereomicroscope, confirming the nodular nature of the lesions, which were often confluent, alternating with empty spaces, giving the tissue a honeycombing aspect. The histological examination at low magnification allowed us to observe the heavy vacuolization of nodular lesions irregularly surrounded by a spongy-like wall. The observation at higher magnification of other sections allowed us to identify intra-myofibrillar cists containing presumptive microsporidian elements. The tissue damage derived from the technological processes and gravity of lesions did not allow a morphological characterization of presumptive protozoans. The molecular examination of the nodular lesions and the analysis of the sequence of an 897 bp fragment of the small subunit 16S rRNA revealed 100% identity with Microsporidium theragrae (GenBank accession number MT928885-89) first isolated from the skeletal muscles of Gadus chalcogrammus specimens from the Sea of Okhotsk. This finding confirms the importance of selecting suppliers and raw materials in the seafood industry, as well as the usefulness of an effective traceability system.

Isolation and Identification of Nematode-Infecting Microsporidia.

Nematodes are naturally infected by the fungal-related pathogen microsporidia. These ubiquitous eukaryotic parasites are poorly understood, despite infecting most types of animals. Identifying novel species of microsporidia and studying them in an animal model can expedite our understanding of their infection biology and evolution. Nematodes present an excellent avenue for pursuing such work, as they are abundant in the environment and many species are easily culturable in the laboratory. The protocols presented here describe how to isolate bacterivorous nematodes from rotting substrates, screen them for microsporidia infection, and molecularly identify the nematode and microsporidia species. Additionally, we detail how to remove environmental contaminants and generate a spore preparation of microsporidia from infected samples. We also discuss potential pitfalls and provide suggestions on how to mitigate them. These protocols allow for the identification of novel microsporidia species, which can serve as an excellent starting point for genomic analysis, determination of host specificity, and infection characterization. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Gathering samples Support Protocol 1: Generating 10× and 40× Escherichia coli OP50 and seeding NGM plates Basic Protocol 2: Microsporidia screening, testing for Caenorhabditis elegans susceptibility, and sample freezing Basic Protocol 3: DNA extraction, PCR amplification, and sequencing to identify nematode and microsporidia species Basic Protocol 4: Removal of contaminating microbes and preparation of microsporidia spores Support Protocol 2: Bleach-synchronizing nematodes.

Identification of long non-coding RNAs in response to microsporidia infection in Silkworm, Bombyx mori.

Microsporidia Nosema bombycis (Nb) is a cellular parasite responsible for pébrine disease in silkworms, significantly impacting the sericulture industry. Long non-coding RNAs (lncRNAs), which are RNA fragments longer than 200 nucleotides, are pivotal in a range of cellular and physiological functions. However, the potential role of silkworm lncRNAs in response to Nb infection remains unknown. This study conducted transcriptome sequencing on both larvae and Nb-infected midguts of silkworms, identifying 1,440 lncRNAs across all examined midgut samples. Within the Nb-infected group, 42 differentially expressed lncRNAs (DElncRNAs) and 305 differentially expressed mRNAs (DEmRNAs) were detected. Functional annotation and pathway analysis showed that these DEmRNAs are mostly involved in metabolism, apoptosis, autophagy, and other key pathways. The co-expression network of DEmRNAs and DElncRNAs illustrates that 1 gene could be regulated by multiple lncRNAs and 1 lncRNA may target multiple genes, indicating that the regulation of lncRNA is intricate and networked. In addition, the DElncRNA-miRNA-mRNA network showed that some DElncRNAs may be involved in the immune response and metabolism through miRNA. Notably, the study observed an increase in lncRNA MSTRG857.1 following Nb infection, which may promote Nb proliferation. These findings offer insights into the complex interplay between insects and microsporidia.