Full-Length Transcriptome Construction and Systematic Characterization of Virulence Factor-Associated Isoforms in Vairimorpha (Nosema) Ceranae.

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 recent revision of the genera Nosema and Vairimorpha (Microsporidia: Nosematidae) was flawed and misleads the bee scientific community.

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.

Winter Hive Debris Analysis Is Significant for Assessing the Health Status of Honeybee Colonies (Apis mellifera).

Honeybee diseases are one of the most significant and most common causes of honeybee colonies' weakness and death. An early diagnosis of subclinical infections is necessary to implement precautionary and control measures. Sampling debris from hive bottom boards is simple, non-invasive, and cheap. In this study, we collected winter debris samples in apiaries located in the continental part of Croatia. We used molecular methods, PCR and qPCR, for the first time to analyze those samples. Laboratory results were compared with the health condition and strength of honeybee colonies at an apiary in spring. Our study successfully identified the presence and quantity of various pathogens, including the presence of Vairimorpha spp. (Nosema spp.), quintefied Paenibacillus larvae, Acute Bee Paralysis Virus (ABPV), Black Queen Cell Virus (BQCV), Deformed Wing Virus (DWV), and Sacbrood Virus (SBV). However, our analysis did not detect Melissococcus plutonius, Crithidia mellificae, Lotmaria passim, and Aethina tumida. Samples of winter debris were also examined for the presence and quantification of the V. destructor mites, and their natural mite fall was observed in spring. Honeybee colonies were simultaneously infected by an average of four to six pathogens. Some observed honeybee colonies developed characteristic symptoms, while others did not survive the winter.

Pathogen- and host-directed pharmacologic strategies for control of Vairimorpha (Nosema) spp. infection in honey bees.

Microsporidia are obligate intracellular parasites of the Fungal Kingdom that cause widespread infections in nature, with important effects on invertebrates involved in food production systems. The two microsporidian species Vairimorpha (Nosema) ceranae (and the less common Vairimorpha (Nosema) apis) can cause individual disease in honey bees and contribute to colony collapse. The efficacy, safety, and availability of fumagillin, the only drug currently approved to treat microsporidia infection in bees, is uncertain. In this review, we will discuss some of the most promising alternative strategies for the mitigation of Vairimorpha spp. with an emphasis on infection by V. ceranae, now the dominant species infecting bees. We will focus on pharmacologic interventions where the mechanism of action is known and examine both pathogen-directed and host-directed approaches. As limiting toxicity to host cells has been especially emphasized in treating bees that are already facing numerous stressors, strategies that disrupt pathogen-specific targets may be especially advantageous. Therefore, efforts to increase the knowledge and tools for facilitating the discovery of such targets and pharmacologic agents directed against them should be prioritized.

Fall Treatment with Fumagillin Contributes to an Overwinter Shift in Vairimorpha Species Prevalence in Honey Bee Colonies in Western Canada.

(1) Background: Microsporidiosis (nosemosis) is an intestinal disorder of adult honey bees caused by the microsporidian pathogens Vairimorpha apis and Vairimorpha ceranae. In Canada, fumagillin is an approved antibiotic used to treat this disease. However, the recommended dosage is based on efficacy studies for V. apis, the native pathogen in European honey bees. Since the detection of V. ceranae in Apis mellifera, V. ceranae became more prevalent in managed European honey bees and seems to have replaced V. apis due to yet unknown reasons. (2) Methods: This colony study investigated the efficacy of fumagillin administered in the fall to colonies infected with both V. apis and V. ceranae and its effects on the Vairimorpha species' prevalence overwinter. Spore loads in control and fumagillin-treated colonies were analysed by microscopy; Vairimorpha species prevalence was determined molecularly and infection and treatment effects on colony productivity were assessed. (3) Results: Fall fumagillin treatment was associated with a temporary reduction in spore load, but there was no difference in spore loads between treated and control colonies the following spring. Interestingly, fumagillin-treated colonies had a significantly greater prevalence of V. ceranae relative to V. apis the following spring, suggesting fumagillin is less effective in controlling V. ceranae.

Monitoring the Season-Prevalence Relationship of Vairimorpha ceranae in Honey Bees (Apis mellifera) over One Year and the Primary Assessment of Probiotic Treatment in Taichung, Taiwan.

Microsporidiosis, which is caused by the pathogen Vairimorpha ceranae, is a prevalent disease in the honey bee (Apis mellifera) and might lead to significant adult honey bee mortality. In this study, we conducted an annual survey of the mature spore load of V. ceranae in the guts of nurse bees and forager bees in the apiary of National Chung Hsing University (NCHU) in Taiwan. The results indicated that, on average, honey bees hosted approximately 2.13 × 106 mature spore counts (MSCs)/bee in their guts throughout the entire year. The highest number of MSCs was 6.28 × 106 MSCs/bee, which occurred in April 2020, and the lowest number of MSCs was 5.08 × 105 MSCs/bee, which occurred in November 2020. Furthermore, the guts of forager bees had significantly higher (>58%) MSCs than those of nurse bees. To evaluate the potential of the probiotic to treat microsporidiosis, the lactic acid bacterium Leuconostoc mesenteroides TBE-8 was applied to honey bee colonies. A significant reduction (>53%) in MSCs following probiotic treatment was observed, indicating the potential of probiotic treatment for managing microsporidiosis. This research provided information on V. ceranae MSCs in the honey bee gut at NCHU in Taiwan and the MSCs' correlation with the annual season. Furthermore, a potential probiotic treatment for microsporidiosis was assessed for future management.

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.

Functional annotation of a divergent genome using sequence and structure-based similarity.

BACKGROUND: Microsporidia are a large taxon of intracellular pathogens characterized by extraordinarily streamlined genomes with unusually high sequence divergence and many species-specific adaptations. These unique factors pose challenges for traditional genome annotation methods based on sequence similarity. As a result, many of the microsporidian genomes sequenced to date contain numerous genes of unknown function. Recent innovations in rapid and accurate structure prediction and comparison, together with the growing amount of data in structural databases, provide new opportunities to assist in the functional annotation of newly sequenced genomes. RESULTS: In this study, we established a workflow that combines sequence and structure-based functional gene annotation approaches employing a ChimeraX plugin named ANNOTEX (Annotation Extension for ChimeraX), allowing for visual inspection and manual curation. We employed this workflow on a high-quality telomere-to-telomere sequenced tetraploid genome of Vairimorpha necatrix. First, the 3080 predicted protein-coding DNA sequences, of which 89% were confirmed with RNA sequencing data, were used as input. Next, ColabFold was used to create protein structure predictions, followed by a Foldseek search for structural matching to the PDB and AlphaFold databases. The subsequent manual curation, using sequence and structure-based hits, increased the accuracy and quality of the functional genome annotation compared to results using only traditional annotation tools. Our workflow resulted in a comprehensive description of the V. necatrix genome, along with a structural summary of the most prevalent protein groups, such as the ricin B lectin family. In addition, and to test our tool, we identified the functions of several previously uncharacterized Encephalitozoon cuniculi genes. CONCLUSION: We provide a new functional annotation tool for divergent organisms and employ it on a newly sequenced, high-quality microsporidian genome to shed light on this uncharacterized intracellular pathogen of Lepidoptera. The addition of a structure-based annotation approach can serve as a valuable template for studying other microsporidian or similarly divergent species.

A Systematic Review of Fumagillin Field Trials for the Treatment of Nosema Disease in Honeybee Colonies.

This article systematically reviews controlled field trials of fumagillin dicyclohexylamine in honeybee colonies to determine whether fumagillin effectively controls nosema and whether it is beneficial to colonies. Fifty publications were found that described controlled field trials of fumagillin in honeybee colonies between 1952 and 2023. Fumagillin consistently reduced the prevalence and severity of nosema infections. Doses applied in recent studies were similar to or below those recommended historically. Furthermore, our study showed no negative effects on colony health. Improvements in colony survival, size, and honey production have been demonstrated frequently, though not consistently, in both historic and recent studies. Nevertheless, some practices are not optimal. Treatment decision thresholds based on the number of spores per bee are not well supported by evidence and may be no better than calendar-based prophylactic treatments. In addition, reasonable recommendations to employ quarantine and disinfection procedures together with fumagillin treatment do not appear to have been widely adopted. When used as stand-alone treatments, both the fall- and spring-label doses provide benefits but may be too low and short-term to ensure full control of the disease.

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.

Commercial probiotic formulas Bactocell and Levucell promote spring brood production in Apis mellifera L. (Hymenoptera: Apidae) colonies.

Honey bees are essential pollinators for several economically important crops. In temperate countries, honey bee colonies face multiple threats during the overwintering period, such as food availability, diseases, and confinement. Beekeepers commonly use chemicals to improve colony health during winter, but these products can have a negative impact on bee health and pathogens can develop resistance to them. Thus, there is a need for further development of alternative treatments. The aim of this study was to evaluate the impact of one endogenic bacterium (Bombella apis) and 2 commercial probiotic formulas (Bactocell and Levucell) on colony survival, spring development, and Vairimorpha (formerly Nosema) spp. spore count. Probiotic treatments were given in 1: 1 sugar syrup in October 2017 and April 2018, once a week for 2 wk. One experimental group was given Fumagilin-B, the only product approved in Canada to prevent nosemosis, once in October. The administration of 2 commercial probiotics, Bactocell (Pediococcus acidilactici) and Levucell (Saccharomyces cerevisiae boulardii), led to a significant increase in the number of sealed brood cells in spring. None of the probiotic treatments impacted the honey bee gut load of Vairimorpha spp. spores. The results suggest that beneficial microorganisms can improve spring development and performance of honey bee colonies.

Comparison of individual and pooled sampling methods for estimation of Vairimorpha (Nosema) spp. levels in experimentally infected honey bee colonies.

The microsporidian pathogens Vairimorpha apis and V. ceranae are known to cause intestinal infection in honey bees and are associated with decreased colony productivity and colony loss. The widely accepted method for determining Vairimorpha colony infection level for risk assessment and antibiotic treatment is based on spore counts of 60 pooled worker bees using light microscopy. Given that honey bee colonies consist of as many as 1,000 times more individuals, the number of bees collected for Vairimorpha detection may significantly impact the estimated colony infection level, especially in the case of uneven distribution of high- and low-infected individuals within a hive. Hence, we compared the frequency and severity of Vairimorpha infection in individual bees to pooled samples of 60, 120, and 180 bees, as well as compared the Vairimorpha spp. prevalence in pooled samples of 60 and 180 bees. Overall, we did not find significant differences in spore counts in pooled samples containing incremental numbers of bees, although we observed that, in less-infected colonies, a low frequency of highly infected individuals influenced the estimated colony infection level. Moreover, Vairimorpha spp. prevalence did not differ significantly among the pooled bee samples tested. Increasing the number of pooled bees from the recommended 60 bees to 180 bees did not yield a more accurate representation of colony infection level for highly infected colonies, but the clinical importance of a low frequency of highly infected individuals in less-infected colonies needs to be addressed in future studies.

Alkaloid content in microsporidia-infected Adalia bipunctata (Coleoptera: Coccinellidae) life stages, and pathogen spore load in adults after exposure to physical stress.

The two-spotted lady beetle, Adalia bipunctata L., displays warning colouration that is reinforced by the production of adaline and adalinine. These alkaloids are thought to provide defense against predation throughout all life stages of A. bipunctata and may play a role in the insect immune system. Vairimorpha (Nosema) adaliae, a microsporidium described from A. bipunctata, has minimal effects on its host (delayed larval development) when reared under optimum conditions but stress factors are shown to affect the development of microsporidiosis. The objectives of this study were to determine the effects of V. adaliae on relative alkaloid content (adaline) during A. bipunctata development, and to evaluate the combined effects of physical stress and infection on adult beetles (relative alkaloid content and infection load). First-instar larvae were isolated from uninfected and V. adaliae-infected colonies. Eggs and first-instar larvae were immediately prepared for alkaloid analysis, whereas late-instar larvae, pupae and adults were systematically processed when each reached their designated developmental stage. Upon eclosion, a subsample of beetles was exposed to varying amounts of physical agitation: control (no shaking), alternate shaking (every other day), and daily shaking. Immediately following these stress trials, alkaloid samples were collected for analysis and spore loads were assessed. Overall, relative adaline proportions increased from egg to adult. Uninfected individuals had significantly higher relative proportions of adaline than did infected individuals during early development; however, adaline content was higher in infected A. bipunctata from the third-instar onwards, when compared to their uninfected counterparts. Following exposure to physical agitation on alternate days, uninfected adults had a significantly higher relative proportion of adaline than did infected adults. Interestingly, exposure to different levels of agitation had no significant effect on alkaloid production for either uninfected or infected beetles. Mean spore counts were significantly higher for adults that were exposed to daily shaking when compared to individuals from the control and alternate shaking groups. From a biological perspective, one would expect to observe differences in alkaloid production through coccinellid development, as each successive life stage faces different external pressures and risks. When infected with the microsporidium V. adaliae, however, adaline production was reduced during early development but increased significantly in late life stages.

Symptomatic Infection with Vairimorpha spp. Decreases Diapause Survival in a Wild Bumble Bee Species (Bombus griseocollis).

Vairimorpha, a microsporidian parasite (previously classified as Nosema), has been implicated in the decline of wild bumble bee species in North America. Previous studies examining its influence on colony performance have displayed variable results, from extremely detrimental effects to no observable influence, and little is known about the effects it has on individuals during the winter diapause, a bottleneck for survival in many annual pollinators. Here, we examined the effect of Vairimorpha infection, body size, and mass on diapause survival in Bombus griseocollis gynes. We demonstrate that gyne survival length in diapause is negatively affected by symptomatic Vairimorpha infection of the maternal colony but does not correlate with individual pathogen load. Our findings further indicate that increased body mass offers a protective effect against mortality during diapause in infected, but not in healthy, gynes. This suggests that access to adequate nutritional resources prior to diapause might offset the harmful effect of Vairimorpha infection.

Infection Prevalence of Microsporidia Vairimorpha (Nosema) spp. in Japanese Bumblebees.

Microsporidia are spore-forming intracellular parasites of various invertebrates and vertebrates. Vairimorpha bombi negatively affects the fitness of bumblebees and its prevalence correlates with declining bumblebee populations. The invasive alien species Bombus terrestris colonized Japan and possibly introduced new parasites. To assess the infection prevalence of V. bombi in Japanese bumblebees and B. terrestris, we investigated V. bombi infections using PCR and microscopy. The prevalence of sporulating V. bombi infections in three Bombus s. str. species/subspecies was low, whereas that of non/low-sporulating Vairimorpha sp. infections in three Diversobombus species/subspecies was high. Invasive B. terrestris showed low prevalence of non/low-sporulating V. bombi infections and shared the same V. bombi haplotype with B. hypocrita found in Hokkaido, where B. terrestris is present, and in Honshu, where B. terrestris is absent. Although V. bombi may have been introduced with B. terrestris colonies imported from Europe, it seems to be originally distributed in Japan. Furthermore, a new Vairimorpha sp. was found in Japanese bumblebee species. V. bombi and Vairimorpha sp. showed different organ and host specificities in bumblebees. There are no reports on the specific effects of other Vairimorpha spp. on bumblebees; further studies are needed to clarify the individual characteristics of Vairimorpha spp.

Effect of Fumagilin-B treatment timing on nosema (Vairimorpha spp.; Microspora: Nosematidae) abundance and honey bee (Hymenoptera: Apidae) colonies under winter management in the Canadian Prairies.

Fumagilin-B is used to treat nosema infection in honey bee colonies; however, it is unclear whether treatment consistently reduces Vairimorpha ceranae (Fries et al.) abundance and improves colony strength and survival in the Canadian Prairies. This study assessed spring and fall fumagillin treatments on nosema abundance, colony strength, and mortality in 2 different beekeeping regions within Alberta, using both indoor and outdoor wintering management at each site. We compared 4 fumagillin treatments: Spring-only, Fall-only, Spring-and-Fall, and Control (no treatment). The spring treatment dose was ~68 mg/colony, whereas the fall treatment dose was 120 or 48 mg/colony, depending on the year. We found that the colonies were infected predominately with V. ceranae, with V. apis (Zander) present only in mixed infections in a subset of colonies. Although treatment in either the spring or fall did reduce nosema abundance in the short term, it did not eliminate the infection, making continued monitoring necessary. Colony strength was improved by spring treatment in some locations but not consistently, possibly due to the treatment timing or low dose. The combined spring and fall treatment increased colony survival over winter in one of 2 yr. Wintering method did not interact with treatment to affect nosema abundance in the spring. There does not appear to be a significant residual benefit of fall treatment as it did not reduce spring nosema abundance or increase colony population. Therefore, spring treatment should be applied to reduce spring V. ceranae abundance rather than relying on residual efficacy from previous fall treatments.

Evaluation of manuka honey on the microsporidian pathogen Vairimorpha (Nosema) adaliae and its host, the two-spotted lady beetle, Adalia bipunctata L. (Coleoptera: Coccinellidae).

Honey is known for its antibacterial and antifungal properties. Manuka honey was examined for its potential to manage the microsporidium Vairimorpha (Nosema) adaliae infecting Adalia bipunctata larvae. Development time for uninfected larvae fed aphids and water was 13.0 ± 0.2 days, which did not differ significantly from larvae fed aphids and manuka honey. Development of V. adaliae-infected larvae fed aphids and water was 16.3 ± 0.5 days, compared to 15.0 ± 0.2, 15.2 ± 0.3, and 15.6 ± 0.2 days for larvae fed aphids and 5 %, 10 %, or 15 % manuka honey, respectively. Development time was shorter for all honey treatments, but only those fed 5 % manuka differed significantly from the control. Control adults had 19.4 ± 3.0 spores/120 µm2, compared to 19.0 ± 2.0, 19.1 ± 2.1, and 14.3 ± 2.2, for adults provided with 5 %, 10 %, and 15 % honey, respectively. Although spore loads did not differ significantly (p > 0.05), lighter infections were observed in the group fed 15 % manuka.

Laboratory Cultivation of Vairimorpha (Nosema) ceranae (Microsporidia: Nosematidae) in Artificially Infected Worker Bees.

Nosemosis type C is a dangerous and widespread disease of the adult European honey bee Apis mellifera and is caused by the spore-forming intracellular parasite Vairimorpha (Nosema) ceranae. The search for new ways of therapy for this disease is complicated due to the seasonal availability of V. ceranae-infected insects as well as the lack of a developed system for the pathogen's cultivation. By carrying out trials which used different infectious dosages of the parasite, spore storage protocols, host age, and incubation temperatures, we present a simple, safe, and efficient method of V. ceranae propagation in artificially infected worker bees in the laboratory. The method is based on feeding the groups of adult worker bees with microsporidian spores and insect maintenance in plastic bottles at 33 °C. The source of the spores originated from the cadavers of infected insects from the previous round of cultivation, in which the infective spores persist for up to six months. An analysis of five independent cultivation rounds involving more than 2500 bees showed that the proposed protocol exploiting the dosage of one million spores per bee yielded over 60 million V. ceranae spores per bee, and most of the spore samples can be isolated from living insects.

Squash bees host high diversity and prevalence of parasites in the northeastern United States.

The squash bee Eucera (Peponapis) pruinosa is emerging as a model species to study how stressors impact solitary wild bees in North America. Here, we describe the prevalence of trypanosomes, microsporidians and mollicute bacteria in E. pruinosa and two other species, Bombus impatiens and Apis mellifera, that together comprise over 97% of the pollinator visitors of Cucurbita agroecosystems in Pennsylvania (United States). Our results indicate that all three parasite groups are commonly detected in these bee species, but E. pruinosa often exhibit higher prevalences. We further describe novel trypanosome parasites detected in E. pruinosa, however it is unknown how these parasites impact these bees. We suggest future work investigates parasite replication and infection outcomes.

A new microsporidian pathogen, Vairimorpha gastrophysae sp. nov., isolated from Gastrophysa viridula (Coleoptera: Chrysomelidae).

Gastrophysa viridula DeGeer 1775, the green dock leaf beetle, belongs to a group of beneficial insects, which can be used as a classical biological control agent against sorrels (Rumex sp., Polygonaceae). Therefore, any infection by pathogenic organisms in this beetle is undesirable. In the present study, a new microsporidian pathogen isolated from G. viridula was identified based on morphological and ultrastructural characteristics, supported with a molecular phylogenetic analysis. Light and transmission electron microscopy studies indicated that the microsporidium was polymorphic throughout its life cycle. Sporulation stages were not all in direct contact with the host-cell cytoplasm. The fresh single diplokaryotic spores of the secondary sporulation cycle had a long narrow morphology, measuring about 5 × 2.1 µm (n = 50). Octospores produced in the secondary sporulation cycle were also observed. Morphological and ultrastructural characteristics of the life cycle stages place it within the genus Vairimorpha. The phylogenetic tree constructed on the 16S rRNA gene sequence analysis supports the morphological and ultrastructural characteristics and indicates that the pathogen is closely related to the Vairimorpha clade of microsporidia. The pathogen is named Vairimorpha gastrophysae sp. nov.