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Background: Microsporidia is an obligate intracellular eukaryote, which is capable of parasitizing vertebrates and invertebrates. Nosema ceranae, which can infect both Apis mellifera and Apis cerana, poses a serious threat and causes heavy losses to the worldwide apiculture. During infection, polar tube, a highly specialized invasion structure, ejected from the spore to deliver the sporoplasm into host cells to cause infection. Although seven different polar tube proteins (PTP1 ~ 7) have been reported from various microsporidia and showed key functions associated with spore invasion and proliferation, no systematic analysis on identification and characterization of polar tube proteins from N. ceranae was found., Methods and Results: The polar tube proteins 2 (NcPTP2) was identified from the total polar tube proteins of N. ceranae by LC_MS/MS and the transcriptional profile was performed by RT-PCR. Sequence characterization analysis revealed that NcPTP2 was rich in lysine and had a signal peptide at the N-terminal. It had 3 potential O-glycosylation sites and 6 potential N-glycosylation sites. 25 phosphorylation sites were found on serine, tyrosine and threonine sites. Sequence alignment analysis revealed that NcPTP2 was homologous and had conserved cysteine residues with PTP2 proteins from other microsporidia. Indirect immunofuorescence analysis (IFA) and Immunoelectron Microscopy analysis (IEM) confirmed that NcPTP2 was localized on the polar tube of the germinated spores. The interaction between NcPTP2 and spore wall protein in N. ceranae indicated its potential function in anchoring and coiling of polar tube in spore., Conclusion: NcPTP2 was the first subcellular localized polar tube protein in N. ceranae and this work could provide an important basis for further analyzing the biological functions of polar tube proteins and uncovering the infection mechanism of N. ceranae to the host cells., Competing Interests: Declarations Conflict of interest No conflicts of interest, financial or otherwise, are declared by the authors. Ethical approval It is not applicable., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Microsporidia are unfriendly microorganisms, and their infections cause considerable damage to economically or environmentally important insects like silkworms and honeybees. Thus, the identification of measures to improve host resistance to microsporidia infections is critically needed. Here, an overexpressed miR-6498-5p transgenic silkworm line was constructed. Importantly, the survival rates and median lethal doses of the transgenic line were clearly higher after infection with Nosema bombycis . H&E staining and RT-qPCR analyses revealed an inhibitory effect on the proliferation of N. bombycis in the transgenic larvae. Metabolomics analysis further revealed the presence of 56 differential metabolites between the two lines. KEGG analysis of these 56 metabolites found that they were involved in various amino acid and vitamin metabolism pathways. Notably, VB6 metabolism was enriched among the metabolites, and the pathway was well known for its involvement in the synthesis, interconversion, and degradation of amino acids. These suggest that miR-6498-5p modifies parasitic environments to inhibit the proliferation of N. bombycis by affecting the host amino acid metabolism. These results demonstrate the potential of microRNAs as biomolecules that can promote resistance to microsporidia and provide new insights and a new approach to generate microsporidia-resistant biological materials.IMPORTANCEMicrosporidia have an extremely wide host range and are capable of infecting a wide variety of insects and vertebrates, including humans, and their lethality to multiple species often poses significant environmental management challenge. Here, we successfully constructed a microsporidium-resistant line in the silkworm, based on the overexpression of miR-6498-5p. Our results strongly support the hypothesis that miR-6498-5p efficiently suppresses the proliferation of Nosema bombycis by regulating the host VB6 metabolism, a key pathway for enzymes involved in amino acid transport and protein metabolism. Our study provides new insights for understanding host anti-pathogen defenses toward microsporidia., Competing Interests: The authors declare no conflict of interest.

South America is populated by a wide range of bumble bee species that represent an important source of biodiversity, supporting pollination services in natural and agricultural ecosystems. These pollinators provide unique specific microbial niches, populated by a wide number of microorganisms such as symbionts, environmental opportunistic bacteria, and pathogens. Recently, it was demonstrated how microbial populations are shaped by trophic resources and environmental conditions but also by anthropogenic pressure, which strongly affects microbes' functionality. This study is focused on the impact of different land uses (natural reserve, agroecosystem, and suburban) on the gut microbiome composition of two South American bumble bees, Bombus pauloensis and Bombus bellicosus. Gut microbial DNA extracted from collected bumble bees was sequenced on the Illumina MiSeq platform and correlated with land use. Nosema ceranae load was analyzed with qPCR and correlated with microbiome data. Significant differences in gut microbiome composition between the two wild bumble bee species were highlighted, with notable variations in α- and β-diversity across study sites. Bombus bellicosus showed a high abundance of Pseudomonas, a genus that includes environmental saprobes, and was found to be the second major taxa populating the gut microbiome, probably indicating the vulnerability of this host to environmental pollution. Pathogen analysis unveils a high prevalence of N. ceranae, with B. bellicosus showing higher susceptibility. Finally, Gilliamella exhibited a negative correlation with N. ceranae, suggesting a potential protective role of this commensal taxon. Our findings underscore the importance of considering microbial dynamics in pollinator conservation strategies, highlighting potential interactions between gut bacteria and pathogens in shaping bumble bee health., (© 2024. The Author(s).)

The genus Vairimorpha was proposed for several species of Nosema in 1976 (Pilley, 1976), almost 70 years after Nosema apis Zander (Zander, 1909). Tokarev and colleagues proposed the redefinition of 17 microsporidian species in four genera, Nosema, Vairimorpha, Rugispora, and Oligosporidium, based on phylogenetic trees of two genetic markers (SSU rRNA and RPB1) (Tokarev et al., 2020). Several issues should invalidate this new classification, leading to the synonymization of Vairimorpha within Nosema., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microsporidia are opportunistic fungal-like pathogens that cause microsporidiosis, which results in significant economic losses and threatens public health. Infection of domesticated silkworms by the microsporidium Nosema bombycis causes pébrine disease, for which this species of microsporidia has received much attention. Research has been conducted extensively on this microsporidium over the past few decades to better understand its infection, transmission, host-parasite interaction, and detection. Several tools exist to study this species including the complete genome sequence of N. bombycis. In addition to the understanding of N. bombycis being important for the silkworm industry, this species has become a model organism for studying microsporidia. Research on biology of N. bombycis will contribute to the development of knowledge regarding microsporidia and potential antimicrosporidia drugs. Furthermore, this will provide insight into the molecular evolution and functioning of other fungal pathogens., (© 2024 International Society of Protistologists.)

Purpose: Nosemosis is a disease that infects both Western honeybees (Apis mellifera L.) and Asian honeybees (Apis cerana) and causes colony losses and low productivity worldwide. In order to control nosemosis, it is important to determine the distribution and prevalence of this disease agent in a particular region. For this purpose, a national study was conducted to assess the prevalence of Nosema ceranae and N. apis throughout Türkiye, to perform network analyses of the parasites, and to determine the presence of nosemosis., Methods: In this study which aimed to assess the prevalence of N. apis and N. ceranae in different colony types and regions where beekeeping is intensive in Türkiye, specimens were collected from hives with no clinical signs., Results: A total of 1194 Western honeybee colonies in 400 apiaries from 40 provinces of Türkiye were examined by microscopic and molecular techniques. Nosemosis was found in all of 40 provinces. The mean prevalence ratio was 64.3 ± 3.0, with 95% CI in apiaries and 40.5 ± 2.9, 95% CI in hives. Nosema ceranae DNA was detected in all of positive hives, while N. ceranae and N. apis co-infection was detected in only four colonies., Conclusion: This study showed that nosemosis has spread to all provinces, and it is common in every region of Türkiye. All of the N. ceranae or N. apis samples examined were 100% identical within themselves. Network analysis showed that they were within largest haplotype reported worldwide., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Vairimorpha (Nosema) ceranae is a single-cellular fungus that obligately infects the midgut epithelial cells of adult honeybees, causing bee microsporidiosis and jeopardizing bee health and production. This work aims to construct the full-length transcriptome of V. ceranae and conduct a relevant investigation using PacBio single-molecule real-time (SMRT) sequencing technology. Following PacBio SMRT sequencing, 41,950 circular consensus (CCS) were generated, and 25,068 full-length non-chimeric (FLNC) reads were then detected. After polishing, 4387 high-quality, full-length transcripts were gained. There are 778, 2083, 1202, 1559, 1457, 1232, 1702, and 3896 full-length transcripts that could be annotated to COG, GO, KEGG, KOG, Pfam, Swiss-Prot, eggNOG, and Nr databases, respectively. Additionally, 11 alternative splicing (AS) events occurred in 6 genes were identified, including 1 alternative 5' splice-site and 10 intron retention. The structures of 225 annotated genes in the V. ceranae reference genome were optimized, of which 29 genes were extended at both 5' UTR and 3' UTR, while 90 and 106 genes were, respectively, extended at the 5' UTR as well as 3' UTR. Furthermore, a total of 29 high-confidence lncRNAs were obtained, including 12 sense-lncRNAs, 10 lincRNAs, and 7 antisense-lncRNAs. Taken together, the high-quality, full-length transcriptome of V. ceranae was constructed and annotated, the structures of annotated genes in the V. ceranae reference genome were improved, and abundant new genes, transcripts, and lncRNAs were discovered. Findings from this current work offer a valuable resource and a crucial foundation for molecular and omics research on V. ceranae .

The current status of Nosema spp. infections in A. mellifera throughout Eurasia was characterized using electronic databases. Although N. ceranae was predominantly detected in southwestern and south-central regions and N. apis in northwestern and north-central areas, most studies reported the occurrence of both species in Eurasia. In addition, the occurrence of Nosema spp. and Ptp3 gene haplotypes was investigated in the Republic of Tatarstan, Russia. Most of the examined honey bees were infected with both N. apis and N. ceranae. N. apis and N. ceranae isolates were either heterozygous or belonged to different strains and showed infection with more than one strain. New haplotypes were found for N. apis and N. ceranae in the Republic of Tatarstan, Russia. This study expands the data regarding existing haplotypes of Nosema species: there are currently 9 shared and 56 unique Ptp3 nucleotide sequence haplotypes of N. ceranae, and 2 shared and 7 unique haplotypes of N. apis, respectively., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Gene editing techniques are widely and effectively used for the control of pathogens, but it is difficult to directly edit the genes of Microsporidia due to its unique spore wall structure. Innovative technologies and methods are urgently needed to break through this limitation of microsporidia therapies. Here, we establish a microsporidia-inducible gene editing system through core components of microsporidia secreted proteins, which could edit target genes after infection with microsporidia. We identified that Nosema bombycis NB29 is a secretory protein and found to interact with itself. The NB29-N3, which lacked the nuclear localization signal, was localized in the cytoplasm, and could be tracked into the nucleus after interacting with NB29-B. Furthermore, the gene editing system was constructed with the Cas9 protein expressed in fusion with the NB29-N3. The system could edit the exogenous gene EGFP and the endogenous gene BmRpn3 after overexpression of NB29 or infection with N. bombycis .

Lipid droplets (LDs) are dynamic organelles that participate in the regulation of lipid metabolism and cellular homeostasis inside of cells. LD-associated proteins, also known as perilipins (PLINs), are a family of proteins found on the surface of LDs that regulate lipid metabolism, immunity, and other functions. In silkworms, pébrine disease caused by infection by the microsporidian Nosema bombycis ( Nb ) is a severe threat to the sericultural industry. Although we found that Nb relies on lipids from silkworms to facilitate its proliferation, the relationship between PLINs and Nb proliferation remains unknown. Here, we found Nb infection caused the accumulation of LDs in the fat bodies of silkworm larvae. The characterized perilipin1 gene ( plin1 ) promotes the accumulation of intracellular LDs and is involved in Nb proliferation. plin1 is similar to perilipin1 in humans and is conserved in all insects. The expression of plin1 was mostly enriched in the fat body rather than in other tissues. Knockdown of plin1 enhanced Nb proliferation, whereas overexpression of plin1 inhibited its proliferation. Furthermore, we confirmed that plin1 increased the expression of the Domeless and Hop in the JAK-STAT immune pathway and inhibited Nb proliferation. Taken together, our current findings demonstrate that plin1 inhibits Nb proliferation by promoting the JAK-STAT pathway through increased expression of Domeless and Hop . This study provides new insights into the complicated connections among microsporidia pathogens, LD surface proteins, and insect immunity.IMPORTANCELipid droplets (LDs) are lipid storage sites in cells and are present in almost all animals. Many studies have found that LDs may play a role in host resistance to pathogens and are closely related to innate immunity. The present study found that a surface protein of insect lipid droplets could not only regulate the morphological changes of lipid droplets but also inhibit the proliferation of a microsporidian pathogen Nosema bombycis ( Nb ) by activating the JAK-STAT signaling pathway. This is the first discovery of the relationship between microsporidian pathogen and insect lipid surface protein perilipin and insect immunity., Competing Interests: The authors declare no conflict of interest.

Honeybees are an indispensable pollinator in nature with pivotal ecological, economic, and scientific value. However, a full-length transcriptome for Apis mellifera , assembled with the advanced third-generation nanopore sequencing technology, has yet to be reported. Here, nanopore sequencing of the midgut tissues of uninoculated and Nosema ceranae -inoculated A. mellifera workers was conducted, and the full-length transcriptome was then constructed and annotated based on high-quality long reads. Next followed improvement of sequences and annotations of the current reference genome of A. mellifera. A total of 5,942,745 and 6,664,923 raw reads were produced from midguts of workers at 7 days post-inoculation (dpi) with N. ceranae and 10 dpi, while 7,100,161 and 6,506,665 raw reads were generated from the midguts of corresponding uninoculated workers. After strict quality control, 6,928,170, 6,353,066, 5,745,048, and 6,416,987 clean reads were obtained, with a length distribution ranging from 1 kb to 10 kb. Additionally, 16,824, 17,708, 15,744, and 18,246 full-length transcripts were respectively detected, including 28,019 nonredundant ones. Among these, 43,666, 30,945, 41,771, 26,442, and 24,532 full-length transcripts could be annotated to the Nr, KOG, eggNOG, GO, and KEGG databases, respectively. Additionally, 501 novel genes (20,326 novel transcripts) were identified for the first time, among which 401 (20,255), 193 (13,365), 414 (19,186), 228 (12,093), and 202 (11,703) were respectively annotated to each of the aforementioned five databases. The expression and sequences of three randomly selected novel transcripts were confirmed by RT-PCR and Sanger sequencing. The 5' UTR of 2082 genes, the 3' UTR of 2029 genes, and both the 5' and 3' UTRs of 730 genes were extended. Moreover, 17,345 SSRs, 14,789 complete ORFs, 1224 long non-coding RNAs (lncRNAs), and 650 transcription factors (TFs) from 37 families were detected. Findings from this work not only refine the annotation of the A. mellifera reference genome, but also provide a valuable resource and basis for relevant molecular and -omics studies.

The genus Serratia harbors opportunistic pathogenic species, among which Serratia marcescens is pathogenic for honeybees although little studied. Recently, virulent strains of S. marcescens colonizing the Varroa destructor mite's mouth were found vectored into the honeybee body, leading to septicemia and death. Serratia also occurs as an opportunistic pathogen in the honeybee's gut with a low absolute abundance. The Serratia population seems controlled by the host immune system, but its presence may represent a hidden threat, ready to arise when honeybees are weakened by biotic and abiotic stressors. To shed light on the Serratia pathogen, this research aims at studying Serratia 's development dynamics in the honeybee body and its interactions with the co-occurring fungal pathogen Vairimorpha ceranae . Firstly, the degree of pathogenicity and the ability to permeate the gut epithelial barrier of three Serratia strains, isolated from honeybees and belonging to different species ( S. marcescens , Serratia liquefaciens , and Serratia nematodiphila ), were assessed by artificial inoculation of newborn honeybees with different Serratia doses (10 4 , 10 6 , and 10 8 cells/mL). The absolute abundance of Serratia in the gut and in the hemocoel was assessed in qPCR with primers targeting the luxS gene. Moreover, the absolute abundance of Serratia was assessed in the gut of honeybees infected with V. ceranae at different development stages and supplied with beneficial microorganisms and fumagillin. Our results showed that all tested Serratia strains could pass through the gut epithelial barrier and proliferate in the hemocoel, with S. marcescens being the most pathogenic. Moreover, under cage conditions, Serratia better proliferates when a V. ceranae infection is co-occurring, with a positive and significant correlation. Finally, fumagillin and some of the tested beneficial microorganisms could control both Serratia and Vairimorpha development. Our findings suggest a correlation between the two pathogens under laboratory conditions, a co-occurring infection that should be taken into consideration by researches when testing antimicrobial compounds active against V. ceranae , and the related honeybees survival rate. Moreover, our findings suggest a positive control of Serratia by the environmental microorganism Apilactobacillus kunkeei in a in vivo model, confirming the potential of this specie as beneficial bacteria for honeybees., Competing Interests: Author DS is/was employed by the company Advance Science Forbairt Eolaiochta Teo Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Braglia, Alberoni, Garrido, Porrini, Baffoni, Scott, Eguaras, Di Gioia and Mifsud.)

Pollinators are vital for food security and the maintenance of terrestrial ecosystems. Bumblebees are important pollinators across northern temperate, arctic, and alpine ecosystems, yet are in decline across the globe. Vairimorpha bombi is a parasite belonging to the fungal class Microsporidia that has been implicated in the rapid decline of bumblebees in North America, where it may be an emerging infectious disease. To investigate the evolutionary basis of pathogenicity of V. bombi, we sequenced and assembled its genome using Oxford Nanopore and Illumina technologies and performed phylogenetic and genomic evolutionary analyses. The genome assembly for V. bombi is 4.73 Mb, from which we predicted 1,870 protein-coding genes and 179 tRNA genes. The genome assembly has low repetitive content and low GC content. V. bombi's genome assembly is the smallest of the Vairimorpha and closely related Nosema genera, but larger than those found in the Encephalitozoon and Ordospora sister clades. Orthology and phylogenetic analysis revealed 18 core conserved single-copy microsporidian genes including the histone acetyltransferase (HAT) GCN5. Surprisingly, V. bombi was unique to the microsporidia in not encoding the second predicted HAT ESA1. The V. bombi genome assembly annotation included 265 unique genes (i.e. not predicted in other microsporidia genome assemblies), 20% of which encode a secretion signal, which is a significant enrichment. Intriguingly, of the 36 microsporidian genomes we analyzed, 26 also had a significant enrichment of secreted signals encoded by unique genes, ranging from 6 to 71% of those predicted genes. These results suggest that microsporidia are under selection to generate and purge diverse and unique genes encoding secreted proteins, potentially contributing to or facilitating infection of their diverse hosts. Furthermore, V. bombi has 5/7 conserved spore wall proteins (SWPs) with its closest relative V. ceranae (that primarily infects honeybees), while also uniquely encoding four additional SWPs. This gene class is thought to be essential for infection, providing both environmental protection and recognition and uptake into the host cell. Together, our results show that SWPs and unique genes encoding a secretion signal are rapidly evolving in the microsporidia, suggesting that they underpin key pathobiological traits including host specificity and pathogenicity., Competing Interests: Conflicts of interest. The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Purpose: Bumblebees are an important group of insects in the pollination of various vegetables, fruits, oilseeds, legumes, and the fodder crops. Compared to honeybees, they have a wider choice of hosts and a longer flight period. These bees are used especially for the pollination of plants in greenhouses and are commercially produced for this purpose. Recently, serious decreases have been occurring in bumblebee populations due to various reasons such as pathogens, and some of species are even threatened with extinction. Due to the worldwide decline in pollinator insects, determining the distribution and prevalence of bumblebee pathogens is of great importance. Therefore, this study was conducted to determine the incidence and prevalence of pathogens in Turkish bumblebee populations and how much of each pathogen was in bumblebee samples., Methods: A total of 172 Bombus terrestris (Linnaeus,1758) samples (21 samples from commercial enterprises, 79 samples from greenhouses and 72 samples from nature) were randomly collected from 3 provinces (Antalya, Mersin and İzmir) where greenhouse cultivation is intensively carried out in Turkey. Eighty-nine of these samples were collected in the spring and eighty-three in the autumn. The presence of four pathogens (Nosema bombi, Crithidia bombi, Apicystis bombi, and Locustacarus buchneri) was investigated by PCR using universal primers., Results: The overall prevalence of Nosema bombi, Crithidia bombi, Apicystis bombi, and Locustacarus buchneri was determined as 7.55%, 9.3%, 11.62%, and 4.65%, respectively. Co-infections (5.81%) were only detected in wild-caught (nature) samples. C. bombi and A. bombi infections were detected at higher rates in the spring samples than in the autumn samples (p < 0.05). There was no significant difference between the spring and autumn samples with respect to the presence of N. bombi and L. buchneri (p > 0.05)., Conclusion: The results obtained could be important in determining the prevalence and spread rates of the bumblebee diseases in Turkey and to determine appropriate protection measures. The information gathered should increase our knowledge about the presence of these pathogens in Turkey and could contribute to improve apiarist's practice. More studies are needed to determine the transmission pathways of these pathogens between the populations. Also, complex pathogen interactions in bumblebee populations should be considered in the future to improve bumblebee health., (© 2023. The Author(s) under exclusive licence to Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

Microsporidia cause disease in many beneficial insects, including honey bees, yet few pathogen control tools are available for protecting these important organisms against infection. Some evidence suggests that microsporidia possess a reduced number of genes encoding DNA repair proteins. We hypothesized that microsporidia would thus be susceptible to treatment with DNA-damaging agents and tested this hypothesis using a novel, rapid method for achieving robust and homogenous experimental infection of large numbers of newly emerged honey bees with one of its microsporidia pathogens, Vairimorpha ( Nosema ) ceranae . In carrying out these experiments, we found this novel V. ceranae inoculation method to have similar efficacy as other traditional methods. We show that the DNA-damaging agent bleomycin reduces V. ceranae levels, with minimal but measurable effects on honey bee survival and increased expression of midgut cellular stress genes, including those encoding SHSP. Increased expression of UpdlC suggests the occurrence of epithelial regeneration, which may contribute to host resistance to bleomycin treatment. While bleomycin does reduce infection levels, host toxicity issues may preclude its use in the field. However, with further work, bleomycin may provide a useful tool in the research setting as a potential selection agent for genetic modification of microsporidia.IMPORTANCEMicrosporidia cause disease in many beneficial insects, yet there are few tools available for control in the field or laboratory. Based on the reported paucity of DNA repair enzymes found in microsporidia genomes, we hypothesized that these obligate intracellular parasites would be sensitive to DNA damage. In support of this, we observed that the well-characterized DNA damage agent bleomycin can reduce levels of the microsporidia Vairimorpha ( Nosema ) ceranae in experimental infections in honey bees. Observation of slightly reduced honey bee survival and evidence of sublethal toxicity likely preclude the use of bleomycin in the field. However, this work identifies bleomycin as a compound that merits further exploration for use in research laboratories as a potential selection agent for generating genetically modified microsporidia., Competing Interests: The authors declare no conflict of interest.

Importance: The multiplex-crRNA CRISPR/Cas12a detection method saves hands-on time, reduces the risk of aerosol pollution, and can be directly applied to detecting silkworms infected with Nosema bombycis . This study provides a new approach for the inspection and quarantine of silkworm pébrine disease in sericulture and provides a new method for the detection of other pathogens., Competing Interests: The authors declare no conflict of interest.

Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription-quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6 h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell's energy resources., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study was done to investigate the effects of thymol, fumagillin, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on Nosema sp. spore load, the expression of vitellogenin ( vg ) and superoxide-dismutase-1 ( sod-1 ) genes and mortality of bees infected with N. ceranae . Five healthy colonies were assigned as the negative control, and 25 Nosema sp. infected colonies were assigned to five treatment groups including: the positive control: no additive to sirup; fumagillin 26.4 mg/L, thymol 0.1 g/L, Api-Bioxal 0.64 g/L and Nose-Go 5.0 g/L sirup. The reduction in the number of Nosema sp. spores in fumagillin, thymol, Api-Bioxal and Nose-Go compared to the positive control was 54, 25, 30 and 58%, respectively. Nosema sp. infection in all infected groups increased ( p  < .05) Escherichia coli population compared to the negative control. Nose-Go had a negative effect on lactobacillus population compared to other substances. Nosema sp. infection decreased vg and sod-1 genes expression in all infected groups compared to the negative control. Fumagillin and Nose-Go increased the expression of vg gene, and Nose-Go and thymol increased the expression of sod-1 gene than the positive control. Nose-Go has the potential to treat nosemosis if the necessary lactobacillus population is provided in the gut.

The microsporidian genus Nosema is primarily known to infect insects of economic importance stimulating high research interest, while other hosts remain understudied. Nosema granulosis is one of the formally described Nosema species infecting amphipod crustaceans, being known to infect only two host species. Our first aim was to characterize Nosema spp. infections in different amphipod species from various European localities using the small subunit ribosomal DNA (SSU) marker. Second, we aimed to assess the phylogenetic diversity, host specificity and to explore the evolutionary history that may explain the diversity of gammarid-infecting Nosema lineages by performing a phylogenetic reconstruction based on RNA polymerase II subunit B1 (RPB1) gene sequences. For the host species Gammarus balcanicus, we also analyzed whether parasites were in excess in females to test for sex ratio distortion in relation with Nosema infection. We identified Nosema spp. in 316 individuals from nine amphipod species being widespread in Europe. The RPB1-based phylogenetic reconstruction using newly reported sequences and available data from other invertebrates identified 39 haplogroups being associated with amphipods. These haplogroups clustered into five clades (A-E) that did not form a single amphipod-infecting monophyletic group. Closely related sister clades C and D correspond to Nosema granulosis. Clades A, B and E might represent unknown Nosema species infecting amphipods. Host specificity seemed to be variable with some clades being restricted to single hosts, and some that could be found in several host species. We show that Nosema parasite richness in gammarid hosts is much higher than expected, illustrating the advantage of the use of RPB1 marker over SSU. Finally, we found no hint of sex ratio distortion in Nosema clade A infecting G. balcanicus. This study shows that Nosema spp. are abundant, widespread and diverse in European gammarids. Thus, Nosema is as diverse in aquatic as in terrestrial hosts., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bacela-Spychalska et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Nosema ceranae infects midgut epithelial cells of the Apis species and has jumped from its original host A. cerana to A. mellifera worldwide, raising questions about the response of the new host. We compared the responses of these two species to N. ceranae isolates from A. cerana , A. mellifera from Thailand and A. mellifera from France. Proteomics and transcriptomics results were combined to better understand the impact on the immunity of the two species. This is the first combination of omics analyses to evaluate the impact of N. ceranae spores from different origins and provides new insights into the differential immune responses in honeybees inoculated with N. ceranae from original A. cerana . No difference in the antimicrobial peptides (AMPs) was observed in A. mellifera , whereas these peptides were altered in A. cerana compared to controls. Inoculation of A. mellifera or A. cerana with N. ceranae upregulated AMP genes and cellular-mediated immune genes but did not significantly alter apoptosis-related gene expression. A. cerana showed a stronger immune response than A. mellifera after inoculation with different N. ceranae isolates. N. ceranae from A. cerana had a strong negative impact on the health of A. mellifera and A. cerana compared to other Nosema isolates.

Nosema ceranae is an intracellular parasite invading the midgut of honeybees, which causes serious nosemosis implicated in honeybee colony losses worldwide. The core gut microbiota is involved in protecting against parasitism, and the genetically engineering of the native gut symbionts provides a novel and efficient way to fight pathogens. Here, using laboratory-generated bees mono-associated with gut members, we find that Snodgrassella alvi inhibit microsporidia proliferation, potentially via the stimulation of host oxidant-mediated immune response. Accordingly, N. ceranae employs the thioredoxin and glutathione systems to defend against oxidative stress and maintain a balanced redox equilibrium, which is essential for the infection process. We knock down the gene expression using nanoparticle-mediated RNA interference, which targets the γ-glutamyl-cysteine synthetase and thioredoxin reductase genes of microsporidia. It significantly reduces the spore load, confirming the importance of the antioxidant mechanism for the intracellular invasion of the N. ceranae parasite. Finally, we genetically modify the symbiotic S. alvi to deliver dsRNA corresponding to the genes involved in the redox system of the microsporidia. The engineered S. alvi induces RNA interference and represses parasite gene expression, thereby inhibits the parasitism significantly. Specifically, N. ceranae is most suppressed by the recombinant strain corresponding to the glutathione synthetase or by a mixture of bacteria expressing variable dsRNA. Our findings extend our previous understanding of the protection of gut symbionts against N. ceranae and provide a symbiont-mediated RNAi system for inhibiting microsporidia infection in honeybees., (© 2023. The Author(s).)

Microsporidians (Microsporidia) are a diverse group of obligate and intracellular parasites of eukaryotes. There is evidence that the real species diversity in the phylum could be greatly underestimated, especially for microsporidians parasitic on invertebrates. Mosquitoes (Culicidae) are among very important microsporidian host groups. However, to date, no extensive survey on the prevalence of microsporidians in European mosquitoes has been performed. Here, we used mosquitoes collected in west-central Poland and a metabarcoding approach to examine the prevalence and diversity of microsporidian species among European mosquitoes. We found that up to one-third of mosquitoes in Europe may be infected with at least 13 microsporidian species belonging to the genera Amblyospora, Hazardia, Encephalitozoon, Enterocytospora, and Nosema and the holding genus Microsporidium. The lack of a difference in microsporidian prevalence between mosquito sexes implies that other factors, e.g., temperature or humidity, affect microsporidian occurrence in adult mosquitoes. Each microsporidian species was found in at least three mosquito species, which suggests that these microsporidians are polyxenic rather than monoxenic parasites. The co-occurrence of at least two different microsporidian species was found in 3.6% of host individuals. The abundance of microsporidian DNA sequences suggests interactions between co-occurring parasites; however, these results should be confirmed by microscopic and quantitative methods. In addition, further histological research is required to describe Microsporidium sp. PL01 or match its DNA to that of an already described species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The main function of Sec61 complex is participating in the transport of polypeptide chains across the endoplasmic reticulum. The Sec61α subunit is the largest subunit of the Sec61 complex and shows high degree of conservation. In this study, we identified the NbSec61α and NbSec61γ genes in the microsporidian Nosema bombycis for the first time. Multiple sequence alignment showed that the sequence similarity between NbSec61α and homologous proteins of other microsporidia was greater than 48 %. NbSec61α contains a "plug" domain (amino acids 40-74) unique to the Sec61/SecY complex. Phylogenetic analysis based on NbSec61α and NbSec61γ indicated that the N. bombycis was closely related to Nosema granulosis, Nosema ceranae and Nosema apis. Indirect immunfluorescence assay showed that NbSec61α and NbSec61γ were mainly distributed in the perinuclear region of N. bombycis in different developmental phases. qRT-PCR results revealed that the expression level of NbSec61α gene increased in the early stage and reached the highest at 48 h, then decreased in the late stages. After knockdown of NbSec61α, the expression of NbSec61α, NbSec61γ and NbssrRNA genes were all significantly down-regulated. These results suggest that the NbSec61α and NbSec61γ may play an important role in the intracellular development of N. bombycis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Microsporidia are ubiquitous in the environment, infecting almost all invertebrates, vertebrates, and some protists. The microsporidian Nosema bombycis causes silkworms pébrine disease and leads to huge economic losses. Parasite secreted proteins play vital roles in pathogen-host interactions. Serine protease inhibitors (serpins), belonging to the largest and most broadly distributed protease inhibitor superfamily, are also found in Microsporidia. In this study, we characterized 19 serpins (NbSPNs) in N. bombycis ; eight of them were predicted with signal peptides. All NbSPN proteins contain a typical conserved serpin (PF00079) domain. The comparative genomic analysis revealed that microsporidia serpins were only found in the genus Nosema . In addition to N. bombycis , a total of 34 serpins were identified in another six species of Nosema including N. antheraeae (11), N. granulosis (8), Nosema sp. YNPr (3), Nosema sp. PM-1 (3), N. apis (4), and N. ceranae (5). Serpin gene duplications in tandem obviously occurred in Nosema antheranae . Notably, the NbSPNs were phylogenetically clustered with serpins from the Chordopoxvirinae , the subfamily of Poxvirus. All 19 NbSPN transcripts were detected in the infected midgut and fat body, while 19 NbSPN genes except for NbSPN12 were found in the transcriptome of the infected silkworm embryonic cell line BmE-SWU1. Our work paves the way for further study of serpin function in microsporidia.

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis . This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or "camelization" of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs.

SWPs are the major virulence component of microsporidian spores. In microsporidia, SWPs can be found either in exospore or endospore to serve as a putative virulence factor for host cell invasion. SWP5 is a vital protein that involves in exospore localization and supports the structural integrity of the spore wall and this action potentially modulates the course of infection in N. bombycis. Here we report recombinant SWP5 purification using Ni-NTA IMAC and SEC. GFC analysis reveals SWP5 to be a monomer which correlates with the predicted theoretical weight and overlaps with ovalbumin peak in the chromatogram. The raised polyclonal anti-SWP5 antibodies was confirmed using blotting and enterokinase cleavage experiments. The resultant fusion SWP5 and SWP5 in infected silkworm samples positively reacts to anti-SWP5 antibodies is shown in ELISA. Immunoassays and Bioinformatic analysis reveal SWP5 is found to be localized on exospore and this action could indicate the probable role of SWP5 in host pathogen interactions during spore germination and its contribution to microsporidian pathogenesis. This study will support development of a field-based diagnostic kit for the detection N. bombycis NIK-1S infecting silkworms. The analysis will also be useful for the formulation of drugs against microsporidia and pebrine disease., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nosema ceranae is a honey bee gut parasite that has recently spilled to another honey bee host through trading. The impact of infection on the native host is minor, which is substantial in the novel host. In this study, artificial inoculation simulated the parasite transmission from the native to the novel host. We found that the parasite initiated proliferation earlier in the novel host than in the native host. Additionally, parasite gene expression was significantly higher when infecting the novel host compared with the native host, leading to a significantly higher number of spores. Allele frequencies were similar for spores of parasites infecting both native and novel hosts. This suggests that the high number of spores found in the novel host was not caused by a subset of more fit spores from native hosts. Native hosts also showed a higher number of up-regulated genes in response to infection when compared with novel hosts. Our data further showed that native hosts suppressed parasite gene expression and arguably sacrificed cells to limit the parasite. The results provide novel insights into host defenses and gene selection during a parasite spillover event., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wei, Evans, Chen and Huang.)

Among stressors affecting bee health, Nosema microsporidia are prevalent intracellular parasites. Nosema apis and Nosema ceranae have been described in honey bees (Apis spp.), while Nosema bombi has been described in bumble bees (Bombus spp.). Although available molecular methods serve as a complement to microscopic diagnosis of nosemosis, they do not enable accurate quantification of these three Nosema species. We developed three quantitative real-time PCRs (qPCRs) starting from in silico design of specific primers, probes, and recombinant plasmids, to target the RNA polymerase II subunit B1 (RPB1) gene in the three species. The complete methods, including bee grinding, DNA purification, and qPCR, were validated in honey bee (Apis mellifera) homogenate. Specificity was assessed in silico and in vitro with several types of bee samples. The limit of detection was estimated at 4 log 10 copies/honey bee. A small, systematic method bias was corrected for accurate quantification up to 10 log 10 copies/honey bee. Method accuracy was also verified in bumble bee (Bombus terrestris) and mason bee (Osmia bicornis) homogenates in the range of 5 to 10 log 10 copies/bee. These validated qPCR methods open perspectives in nosemosis diagnosis and in the study of the parasite's eco-dynamics in managed and wild bees., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Purpose: Pebrine as the most dangerous disease of silkworm mostly caused by Nosema species has caused huge economic losses. There is no information on the species and the genomic sequences of the pebrine-causing microsporidia in Iran., Methods: In the present research, we tried to determine the sequences of two regions of rDNA using molecular methods. First, infected larvae and mother moths were collected from several farms in the north of Iran for identification and molecular characterization of microsporidian isolates. After extracting the spores and genomic DNA from the collected samples, two fragments of internal transcribed spacer (ITS) rDNA and small subunit (SSU) rDNA were amplified and sequenced, and registered in NCBI database and then, the phylogenetic tree was drawn., Results: Results showed the obtained sequences (ITS rDNA: Accession No. MZ322002 and SSU rDNA: Accession No. MZ314703) represent a new strain of Nosema bombycis, which differs from the sequences deposited in the NCBI., Conclusion: The new N. bombycis strain identified in our study will help in control and management of the pebrine disease by specific detection of the infectious agent., (© 2022. The Author(s) under exclusive licence to Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

Microsporidia are obligate intracellular parasites and possess a unique way of invading hosts, namely germination. Microsporidia are able to infect almost all animal cells by germination. During the process, the polar tube extrudes from the spores within, thus injecting infectious sporoplasm into the host cells. Previous studies indicated that subtilisin-like protease 1 (NbSLP1) of microsporidia Nosema bombycis were located at the polar cap of germinated spores where the polar tube extrusion. We hypothesized that NbSLP1 is an essential player in the germination process. Normally, SLP need to be activated by autoproteolysis under conditions. In this study, we found that the signal peptide of NbSLP1 affected the activation of protease, two self-cleavage sites were involved in NbSLP1 maturation between Ala 104 Asp 105 and Ala 124 Asp 125 respectively. Mutants at catalytic triad of NbSLP1 confirmed the decreasing of autoproteolysis. This study demonstrates that intramolecular proteolysis is required for NbSLP1 maturation. The protease undergoes a series of sequential N-terminal cleavage events to generate the mature enzyme. Like other subtilisin-like enzymes, catalytic triad of NbSLP1 are significant for the self-activation of NbSLP1. In conclusion, clarifying the maturation of NbSLP1 will be valuable for understanding the polar tube ejection mechanism of germination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Li, Liu, Dang, Sun, Sheng, Hu, Bao, Chen, Pan and Zhou.)

As a typical species of microsporidium, Nosema bombycis is the pathogen causing the pébrine disease of silkworm. Rapid proliferation of N. bombycis in host cells requires replication of genetic material. As eukaryotic origin recognition protein, origin recognition complex (ORC) plays an important role in regulating DNA replication, and Orc1 is a key subunit of the origin recognition complex. In this study, we identified the Orc1 in the microsporidian N. bombycis (NbOrc1) for the first time. The NbOrc1 gene contains a complete ORF of 987 bp in length that encodes a 328 amino acid polypeptide. Indirect immunofluorescence results showed that NbOrc1 were colocalized with Nbactin and NbSAS-6 in the nuclei of N. bombycis. Subsequently, we further identified the interaction between the NbOrc1 and Nbactin by CO-IP and Western blot. These results imply that Orc1 may be involved in the proliferation of the microsporidian N. bombycis through interacting with actin., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Insect pollination is crucial for the maintenance of natural and managed ecosystems but the functioning of this ecosystem service is threatened by a worldwide decline of pollinators. Key factors in this situation include the spread and interspecific transmission of pathogens worldwide through the movement of managed pollinators. Research on this field has been mainly conducted in some particular species, while studies assessing the interspecific transmission of pathogens at a community level are scarce. However, this information is pivotal to design strategies to protect pollinators. Herein, we analysed the prevalence of two common microsporidia pathogens of managed honey bees (Nosema ceranae and N. apis) in bee communities of semiarid Mediterranean areas from the Southeast of the Iberian Peninsula. Our results confirm the ability of N. ceranae to disperse across wild bee communities in semiarid Mediterranean ecosystems since it was detected in 36 Apoidea species (39% of the sampling; for the first time in nine genera). The prevalence of the pathogen did not show any phylogenetic signal which suggests a superfamily host range of the pathogen or that wild bees may be acting only as vectors of N. ceranae. In addition, N. apis was detected in an Eucera species, which is the second time it has been detected by molecular techniques in a host other than the honey bee. Our study represents the primary assessment of the prevalence of microsporidia at community level in Mediterranean areas and provides outstanding results on the ability of Nosema pathogens to spread across the landscape., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

CCT is a chaperonin which is widely present in eukaryotic cells and mainly involves in the folding and assembly of cytoskeletal proteins β-tubulin and actin. The alpha subunit of CCT(CCTα) plays a pivotal role in the folding and assembly of cytoskeletal protein(s) as an individuals or complexes. In this study, we report cloning, characterization and expression of the CCTα of Nosema bombycis (NbCCTα) for the first time. The NbCCTα gene contains a complete ORF of 1629 bp in length that encodes a 542-amino acid polypeptide. The NbCCTα is 59.662 kDa molecular weight in size with an isoelectric point (pI) of 5.81, no signal peptide or transmembrane domain. The IFA results showed that the NbCCTα was co-localized with actin and β-tubulin in the cytoplasm, nucleus, nuclear membrane and plasma membrane of N. bombycis in the process of proliferation. qPCR analysis showed that the relative expression level of NbCCTα increased from 24 h to 96 h post-infection (hp.i) of N. bombycis, and reached the highest at 96 hp.i. The relative expression level of NbCCTα gene after RNAi was restrained at a low level from 48 hp.i to 96 hp.i. Knockdown of NbCCTα gene down-regulated the expression of Nbβ-tubulin and Nbactin genes. These results imply that NbCCTα may play an important role in the lifecycle of N. bombycis., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Microsporidia are obligate intracellular parasites that can infect a wide range of vertebrates and invertebrates including humans and insects, such as silkworm and bees. The microsporidium Nosema bombycis can cause pebrine in Bombyx mori , which is the most destructive disease in the sericulture industry. Although membrane proteins are involved in a wide range of cellular functions and part of many important metabolic pathways, there are rare reports about the membrane proteins of microsporidia up to now. We screened a putative membrane protein Ycf 1 from the midgut transcriptome of the N. bombycis -infected silkworm. Gene cloning and bioinformatics analysis showed that the Ycf 1 gene contains a complete open reading frame (ORF) of 969 bp in length encoding a 322 amino acid polypeptide that has one signal peptide and one transmembrane domain. Indirect immunofluorescence results showed that Ycf 1 protein is distributed on the plasma membrane. Expression pattern analysis showed that the Ycf 1 gene expressed in all developmental stages of N. bombycis . Knockdown of the Ycf 1 gene by RNAi effectively inhibited the proliferation of N. bombycis . These results indicated that Ycf 1 is a membrane protein and plays an important role in the life cycle of N. bombycis ., Competing Interests: The authors declare there are no competing interests., (©2022 Chen et al.)

Nosema ceranae is an intracellular parasite that infects honeybees' gut altering the digestive functions; therefore, it has the potential of affecting the composition of the gut microbiome. In this work, individual bees of known age were sampled both in spring and autumn, and their digestive tracts were assessed for N. ceranae infection. Intestinal microbiome was assessed by sequencing the bacterial 16S rRNA gene in two different gut sections, the anterior section (AS; midgut and a half of ileum) and the posterior section (PS; second half of ileum and rectum). A preliminary analysis with a first batch of samples (n = 42) showed that AS samples had a higher potential to discriminate between infected and non-infected bees than PS samples. As a consequence, AS samples were selected for subsequent analyses. When analyzing the whole set of AS samples (n = 158) no changes in α- or β-diversity were observed between infected and non-infected bees. However, significant changes in the relative abundance of Proteobacteria and Firmicutes appeared when a subgroup of highly infected bees was compared to the group of non-infected bees. Seasonality and bees' age had a significant impact in shaping the bacteriome structure and composition of the bees' gut. Further research is needed to elucidate possible associations between the microbiome and N. ceranae infection in order to find efficient strategies for prevention of infections through modulation of bees' microbiome., (© 2022. The Author(s).)

Background: Microsporidia, a group of obligate intracellular fungal-related parasites, have been used as efficient biocontrol agents for agriculture and forestry pests due to their host specificity and transovarial transmission. They mainly infect insect pests through the intestinal tract, but the interactions between microsporidia and the gut microbiota of the host have not been well demonstrated., Results: Based on the microsporidia-Bombyx mori model, we report that the susceptibility of silkworms to exposure to the microsporidium Nosema bombycis was both dose and time dependent. Comparative analyses of the silkworm gut microbiome revealed substantially increased abundance of Enterococcus belonging to Firmicutes after N. bombycis infection. Furthermore, a bacterial strain (LX10) was obtained from the gut of B. mori and identified as Enterococcus faecalis based on 16S rRNA sequence analysis. E. faecalis LX10 reduced the N. bombycis spore germination rate and the infection efficiency in vitro and in vivo, as confirmed by bioassay tests and histopathological analyses. In addition, after simultaneous oral feeding with E. faecalis LX10 and N. bombycis, gene (Akirin, Cecropin A, Mesh, Ssk, DUOX and NOS) expression, hydrogen peroxide and nitric oxide levels, and glutathione S-transferase (GST) activity showed different degrees of recovery and correction compared with those under N. bombycis infection alone. Finally, the enterococcin LX protein was identified from sterile LX10 fermentation liquid based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis., Conclusion: Altogether, the results revealed that E. faecalis LX10 with anti-N. bombycis activity might play an important role in protecting silkworms from microsporidia. Removal of these specific commensal bacteria with antibiotics and utilization of transgenic symbiotic systems may effectively improve the biocontrol value of microsporidia. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Host-parasite co-evolution is a process of reciprocal, adaptive genetic change. In natural conditions, parasites can shift to other host species, given both host and parasite genotypes allow this. Even though host-parasite co-evolution has been extensively studied both theoretically and empirically, few studies have focused on parasite gene flow between native and novel hosts. Nosema ceranae is a native parasite of the Asian honey bee Apis cerana, which infects epithelial cells of mid-guts. This parasite successfully switched to the European honey bee Apis mellifera, where high virulence has been reported. In this study, we used the parasite N. ceranae and both honey bee species as model organisms to study the impacts of two-host habitat sharing on parasite diversity and virulence. SNVs (Single Nucleotide Variants) were identified from parasites isolated from native and novel hosts from sympatric populations, as well as novel hosts from a parapatric population. Parasites isolated from native hosts showed the highest levels of polymorphism. By comparing the parasites isolated from novel hosts between sympatric and parapatric populations, habitat sharing with the native host significantly enhanced parasite diversity, suggesting there is continuing gene flow of parasites between the two host species in sympatric populations., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The microsporidia Nosema ceranae is an intracellular parasite of honeybees' midgut, highly prevalent in Apis mellifera colonies for which important epidemiological information is still unknown. Our research aimed at understanding how age and season influence the onset of infection in honeybees and its development in the colony environment. Adult worker honeybees of less than 24h were marked and introduced into 6 different colonies in assays carried out in spring and autumn. Bees of known age were individually analyzed by PCR for Nosema spp. infection and those resulting positive were studied to determine the load by Real Time-qPCR. The age of onset and development of infection in each season was studied on a total of 2401 bees and the probability and the load of infection for both periods was established with two statistical models. First N. ceranae infected honeybees were detected at day 5 post emergence (p.e.; spring) and at day 4 p.e. (autumn) and in-hive prevalence increased from that point onwards, reaching the highest mean infection on day 18 p.e. (spring). The probability of infection increased significantly with age in both periods although the age variable better correlated in spring. The N. ceranae load tended to increase with age in both periods, although the age-load relationship was clearer in spring than in autumn. Therefore, age and season play an important role on the probability and the development of N. ceranae infection in honeybees, bringing important information to understand how it spreads within a colony., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jabal-Uriel, Albarracín, Calatayud, Higes and Martín-Hernández.)

There has been increased focus on the role of microbial attack as a potential cause of recent declines in the health of the western honey bee, Apis mellifera. The Nosema species, N. apis and N. ceranae, are microsporidian parasites that are pathogenic to honey bees, and infection by these species has been implicated as a key factor in honey bee losses. Honey bees infected with both Nosema spp. display significant changes in their biology at the cellular, tissue, and organismal levels impacting host metabolism, immune function, physiology, and behavior. Infected individuals lead to colony dysfunction and can contribute to colony disease in some circumstances. The means through which parasite growth and tissue pathology in the midgut lead to the dramatic physiological and behavioral changes at the organismal level are only partially understood. In addition, we possess only a limited appreciation of the elements of the host environment that impact pathogen growth and development. Critical for answering these questions is a mechanistic understanding of the host and pathogen machinery responsible for host-pathogen interactions. A number of approaches are already being used to elucidate these mechanisms, and promising new tools may allow for gain- and loss-of-function experiments to accelerate future progress., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The microsporidium Nosema bombycis can infect and transmit both vertically and horizontally in multiple lepidopteran insects including silkworms and crop pests. While there have been several studies on the N. bombycis spore, there have been only limited studies on the N. bombycis sporoplasm. This chapter reviews what is known about this life cycle stage as well as published studies on purification of the N. bombycis sporoplasm and its survival in an in vitro cell culture system. Genetic transformation techniques have revolutionized the study of many pathogenic organisms. While progress has been made on the development of such systems for microsporidia, this critical problem has not been solved for these pathogens. This chapter provides a summary of the latest research progress on genetic manipulation of N. bombycis., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Nosemosis caused by Vairimorpha ceranae is one of the most important threats to honeybee colonies worldwide. This study aimed to determine the prevalence and intensity of Vairimorpha infection in different types of colonies and locations in Iran. In October 2017 and May 2018, 376 colonies from 97 apiaries were selected for each month according to a randomly clustered design. By considering 3-5 colonies for each apiary, 20 adult bees as pooled samples were collected from each colony. In microscopic analysis, 46.52% and 46.1% of samples in May and October showed Vairimorpha spores, respectively. The infection intensities in May and October were 5.94 ± 0.19 (× 10 6 ) and 5.86 ± 0.23 (× 10 6 ) spores/bee in a pooled sample, respectively. The mean infection intensity ranged from 1.8 to 12.5 (× 10 6 ) spores/bee. Statistically, there were no significant differences in the prevalence and intensity of V. ceranae infection between May and October samples. No significant differences were found among the prevalence rates of infection in the types of colonies; however, the intensity was significantly higher in migratory and mountainous colonies in May and only in migratory colonies in October. There was a significant correlation between the prevalence and intensity of V. ceranae infection (r 2  = 0.695). PCR analysis showed that the samples were only infected with V. ceranae. No intraspecific variation to V. ceranae was found by direct sequencing of the amplified fragment of 16S rRNA. The obtained sequence was mainly 100% similar to those of V. ceranae isolates from European countries., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The fungus Nosema bombycis causes significant economic losses via parasitism of an economically important insect. MicroRNAs (miRNAs) play important roles in regulating host and parasite gene expression via mRNA degradation or by inhibiting protein translation. To investigate whether microRNA-like RNAs (milRNAs) regulate N. bombycis pathogenesis and to better understand the regulatory mechanisms underlying infection, we constructed small RNA libraries from N. bombycis hyphae during the schizont proliferation period. Eleven novel milRNAs were determined by RNA sequencing and stem-loop reverse transcriptase PCR (RT-PCR) assays. Moreover, a virulence-associated milRNA, Nb-milR8, was identified as critical for N. bombycis proliferation by binding and downregulating expression of its target gene, BmPEX16 , in the host during infection. Silencing of Nb-milR8 or overexpression of the target BmPEX16 gene resulted in increased susceptibility of Bombyx mori to N. bombycis infection. Taken together, these results suggest that Nb-milR8 is an important virulence factor that acts as an effector to suppress host peroxidase metabolism, thereby facilitating N. bombycis proliferation. These results provide important novel insights into interactions between pathogenic fungi and their hosts. IMPORTANCE A thorough understanding of fungal pathogen adaptations is essential for treating fungal infections. Recent studies have suggested that the role of small RNAs expressed in fungal microsporidia genomes are important for elucidating the mechanisms of fungal infections. Here, we report 11 novel microRNA-like RNAs (milRNAs) from the fungal microsporidium Nosema bombycis and identified NB-milRNAs that adaptively regulate N. bombycis proliferation. In addition, we demonstrate that N. bombycis modulates small RNA (sRNA)-mediated infection by encoding an Nb-miR8 that downregulates the expression of the host peroxidase metabolism protein BmPEX16, which is essential for peroxisome membrane biogenesis and peroxisome assembly. These results significantly contribute to our understanding of the pathogenic mechanisms of fungi, and especially microsporidia, while providing important targets for genetical engineering-based treatment of microsporidia.

Previously, six miRNAs were identified from the microsporidian parasite Nosema ceranae. By taking advantage of the recently updated N. ceranae and honey bee genome assemblies, we re-analyzed the deep sequencing datasets. Three novel miRNAs were identified, which were further validated by plasmid cloning and sequencing. The miRNAs correlated with significantly higher number of genes from the parasite than the host. Our data suggest the parasitic miRNAs are involved in self-regulation during the proliferation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The chaperonin-containing t-complex polypeptide 1 (CCT) is a molecular chaperone protein that is widely present in eukaryotic cytoplasm and can assist in the folding of newly synthesized proteins. The CCT complex consists of eight completely different subunits, among which the δ subunit plays an extremely important role in the folding and assembly of cytoskeleton proteins as an individual or complex with other subunits. In this study, we identified the CCTδ in the microsporidian Nosema bombycis (NbCCTδ) for the first time. The NbCCTδ gene contains a complete ORF of 1497 bp in length that encodes a 498 amino acid polypeptide. NbCCTδ is expressed throughout the entire lifecycle of N. bombycis and rather higher in early stage of proliferation. Indirect immunofluorescence results showed that NbCCTδ was colocalized with actin and β-tubulin during the proliferative and sporogonic phases of N. bombycis. RNA interference down-regulated the expression of the NbCCTδ gene. These results imply that NbCCTδ may participate in cytoskeleton formation and proliferation of N. bombycis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The single-celled pathogen Nosema bombycis, that can infect silkworm Bombyx mori and other lepidoptera including Spodoptera, is the first identified Microsporidia which has diplokaryotic nuclei throughout the life cycle. Septin proteins can form highly ordered filaments, bundles or ring structures related to the cytokinesis in fungi. Here, three septin proteins (NbSeptin1, NbSeptin2 and NbSeptin3) from Nosema bombycis CQ I are described. These proteins, appear to be conserved within the phylum Microsporidia. NbSeptins transcripts were detected throughout the pathogen developmental cycle and were significantly enhanced from second days of infection, which lead to our hypothesis that NbSeptins play a role in merogony. Immunofluorescence assay (IFA) revealed a broad distribution of NbSeptins in meronts and partly co-localization of NbSeptins. Interestingly, in some of meronts, NbSeptin2 and NbSeptin3 showed localization between the nuclei of the diplokaryon. Yeast two-hybrid and co-immunoprecipitation analysis verified that NbSeptins can interact with each other. Our findings suggest that NbSeptins can cooperate in the proliferation stage of Nosema bombycis and contribute towards the understanding of the rols of septins in microsporidia development., (Copyright © 2021 Elsevier Inc. All rights reserved.)

"Pébrine" is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the β-tubulin gene in the pébrine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 × 10 2 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV) <5% for both intra-assay and inter-assay variability. The assay was used to trace experimental N. bombycis infection of silkworm larvae, in the fat body, midgut and ovary tissues, through pupation and metamorphosis to the emerging female moth, and her larval off-spring, confirming the vertical transmission of N. bombycis in silkworms. The TaqMan assay revealed a gradual increase in infection levels in the post-infection samples. The assay is reliable and simple to implement and can be a suitable complement to microscopy for routine diagnostics and surveillance in silkworm egg production centres with appropriate infrastructure., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Honey bees (Apis mellifera) have a vital role as pollinators of various crops in the global food supply. Honeybee colonies in Egypt have recently experienced an unexplained rise in annual loss due to a phenomenon known as Colony Collapse Disorder (CCD). In the current study, honey bees were collected from 14 sites from eight governorates in Egypt. The genetic diversity among the collected honey bee populations was investigated using the mitochondrial DNA cytochrome oxidase subunit I (COI). The amplified COI regions were sequenced, analyzed and aligned with other GenBank entries. The nucleotide variability of the CO1 gene was estimated. Multiple viral, varroa mites as well as Nosema ceranae infections were tested in honey bee populations using conventional and RT-qPCR. Based on sequence analysis of the COI, six clearly separated mitotypes were characterized for the first time in these sites in Egypt. Sequence analyses showed that most mitotypes belonged to the A lineage and are very close to the Egyptian native bees, A. m. lamarckii found in the gene databank (NCBI) with 98% similarity. Low genetic diversity between the collected samples was observed. Our results elucidated the detection of Nosema cerana; deformed wing virus (DWV), kakugo virus (KV), black queen/cell virus (BQCV), Israel acute paralysis virus (IAPV), varroa destructor virus-1 (VDV-1) and VDV-1/DWV virus in all regions under investigation in addition to varroa mites. These findings highlighted the importance to maintain proper quarantine measures as well as identify the spectrum of exogenous infectious agents in healthy hives over time which would help in developing more effective control and treatment programs against honey bee viruses and pathogens to facilitate efficient breeding programs and establish a more booming beekeeping industry., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

As one of the core framework proteins of nuclear pore complex (NPC), nucleoporin Nupl70 acts as a structural adapter between the nucleolus and nuclear pore membrane and maintains the stability of NPC structure through interaction with other proteins. In this study, we identified a Nup170 protein in the microsporidian Nosema bombycis for the first time and named it as NbNup170. Secondary structure prediction showed that the NbNup170 contains α-helices and random coils. The three-dimensional structure of NbNup170 is elliptical in shape. Phylogenetic analysis based on the Nup170 and homologous sequences showed that N. bombycis clustered together with Vairimorpha ceranae and Vairimorpha apis. The immunofluorescence localization results showed that the NbNup170 was located on the plasma membrane of the dormant spore and transferred to the surface of sporoplasm in a punctate pattern when the dormant spore has finished germination, and that NbNup170 was distributed on the nuclear membrane and both sides of the nuclei of early proliferative phase, and only on the nuclear membrane during sporogonic phase in the N. bombycis. qPCR analysis showed that the relative expression level of NbNup170 maintained at a low level from 30 to 78 h post-infection with N. bombycis, then reached the highest at 102 h, while that of NbNup170 was repressed at a very low level throughout its life cycle by RNA interference. These results suggested that NbNup170 protein is involved in the proliferative phase and active during the sporogonic phase of N. bombycis.

Energy metabolism is important for the proliferation of microsporidia in infected host cells, but there is limited information on the host response. The energy metabolism response of silkworm (Bombyx mori) to microsporidia may help manage Nosema bombycis infections. We analyzed differentially expressed genes in the B.mori midgut transcriptome at two significant time points of microsporidia infection. A total of 1448 genes were up-regulated, while 315 genes were down-regulated. A high proportion of genes were involved in the phosphatidylinositol signaling system, protein processing in the endoplasmic reticulum, and glycerolipid metabolism at 48 h post infection (h p.i.), and a large number of genes were involved in the TCA cycle and protein processing at 120 h p.i. These results showed that the early stages of microsporidia infection affected the basic metabolism and biosynthesis processes of the silkworm. Knockout of Bm&#95;nscaf2860&#95;46 (Bombyx mori isocitrate dehydrogenase, BmIDH) and Bm&#95;nscaf3027&#95;062 (Bombyx mori hexokinase, BmHXK) reduced the production of ATP and inhibited microsporidia proliferation. Host fatty acid degradation, glycerol metabolism, glycolysis pathway, and TCA cycle response to microsporidia infection were also analyzed, and their importance to microsporidia proliferation was verified. These results increase our understanding of the molecular mechanisms involved in N. bombycis infection and provide new insights for research on microsporidia control. IMPORTANCE: Nosema bombycis can be vertically transmitted in silkworm eggs. The traditional prevention and control strategies for microsporidia are difficult and time-consuming, and this is a problem in silkworm culture. Research has mainly focused on host gene functions related to microsporidia infection and host immune responses after microsporidia infection. Little is known about the metabolic changes occurring in the host after infection. Understanding the metabolic changes in the silkworm host could aid in the recognition of host genes important for microsporidia infection and growth. We analyzed host metabolic changes and the main participating pathways at two time points after microsporidia infection and screened the microsporidia-dependent host energy metabolism genes BmIDH and BmHXK. The results revealed genes that are important for the proliferation of Nosema bombycis. These results illustrate how microsporidia hijack the host genome for their growth and reproduction., (Copyright © 2021. Published by Elsevier Inc.)

Muga silkworms are often prone to many diseases since, these are non-domesticated and are reared outdoors. Microsporidia, an obligate intracellular pathogen with spore as its active form, causes pebrine disease in these silkworms. The study has attempted to categorise the transcript data of the Nosema obtained from the infected muga silkworm using gene ontology and KEGG pathway studies. A total of 2850 unigene sets were identified out of which 2739 unigenes were placed under biological, cellular as well as molecular function categories based on the gene ontology (GO) terms. 1620 out of these unigenes sets found their orthologous partner in the corresponding Nosema bombycis transcriptome. The unigenes were found to be enriched under organic substance metabolic process, organic cyclic compound binding and intracellular anatomical structure for biological process, molecular function and cellular components respectively. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis indicated majority of the enzymes were found to be mapped under purine and thiamine metabolic pathways, indicating an increase in the energy metabolism required to establish the infection in the silkworms. The putative virulence genes identified in this study are PTP2, PTP3, SWP12 and SWP26 which were found to be expressed in other Nosema species indigenous to India, indicating a probable conservation of these genes, which are primarily involved in establishing host pathogen interactions. The expression of these genes was in detectable levels in the infected silkworm samples. These genes may be validated further through bioassay in order understand their roles in establishing the infection and propagation of the spores. The identified virulence genes may be further targeted to develop diagnostic tools for identification of the pathogen at early stages of infection.