Revealing the genome of the microsporidian Vairimorpha bombi, a potential driver of bumble bee declines in North America.

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.

Bleomycin reduces Vairimorpha (Nosema) ceranae infection in honey bees with some evident host toxicity.

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.

Harnessing multiplex crRNA enables an amplification-free/CRISPR-Cas12a-based diagnostic methodology for Nosema bombycis.

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.

Functional characterization of Nosema bombycis (microsporidia) trehalase 3.

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.

Widespread infection, diversification and old host associations of Nosema Microsporidia in European freshwater gammarids (Amphipoda).

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.

Proteomics and Immune Response Differences in Apis mellifera and Apis cerana Inoculated with Three Nosema ceranae Isolates.

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.

Engineered symbiotic bacteria interfering Nosema redox system inhibit microsporidia parasitism in honeybees.

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.

The effects of thymol, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on viability, the Nosema sp. spore load and the expression of vg and sod-1 genes in infected honey bees.

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.

Microsporidians (Microsporidia) parasitic on mosquitoes (Culicidae) in central Europe are often multi-host species.

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.

Identification and subcellular colocalization of protein transport protein Sec61α and Sec61γ in Nosema bombycis.

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.

Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth.

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 ß-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.

Specific detection and quantification of three microsporidia infecting bees, Nosema apis, Nosema ceranae, and Nosema bombi, using probe-based real-time PCR.

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 log10 copies/honey bee. A small, systematic method bias was corrected for accurate quantification up to 10 log10 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 log10 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.

Cloning, Expression and Characterization of Spore Wall Protein 5 (SWP5) of Indian Isolate NIK-1S of Nosema bombycis.

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.

Spillover and genome selection of the gut parasite Nosema ceranae between honey bee species.

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.

Maturation of subtilisin-like protease NbSLP1 from microsporidia Nosema bombycis.

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 Ala104Asp105 and Ala124Asp125 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.

Identification and subcellular localization analysis of membrane protein Ycf 1 in the microsporidian Nosema bombycis.

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.

Molecular Characterization of a New Nosema bombycis Strain Detected in Iranian Silkworm Farms.

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.

Effect of Nosema ceranae infection and season on the gut bacteriome composition of the European honeybee (Apis mellifera).

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 ß-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.

Advances in the Genetic Manipulation of Nosema bombycis.

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.

Nosema apis and N. ceranae Infection in Honey bees: A Model for Host-Pathogen Interactions in Insects.

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.