Age and Season Effect the Timing of Adult Worker Honeybee Infection by Nosema ceranae

Nosema ceranae is a microsporidian parasite that threatens current apiculture. N. ceranae-infected honey bees (Apis mellifera) exhibit morbid physiological impairments and reduced honey production, malnutrition, shorter life span, and higher mortality than healthy honey bees. In this study, we found that dimethyl sulfoxide (DMSO) could enhance the survival rate of N. ceranae-infected honey bees. Therefore, we investigated the effect of DMSO on N. ceranae-infected honey bees using comparative RNA sequencing analysis. Our results revealed that DMSO was able to affect several biochemical pathways, especially the metabolic-related pathways in N. ceranae-infected honey bees. Based on these findings, we conclude that DMSO may be a useful alternative for treating N. ceranae infection in apiculture.

The microsporidium Nosema ceranae is a high prevalent parasite of the European honey bee (Apis mellifera). This parasite is spreading across the world into its novel host. The developmental process, and some mechanisms of N. ceranae-infected honey bees, has been studied thoroughly; however, few studies have been carried out in the mechanism of gene expression in N. ceranae during the infection process. We therefore performed the suppressive subtractive hybridization (SSH) approach to investigate the candidate genes of N. ceranae during its infection process. All 96 clones of infected (forward) and non-infected (reverse) library were dipped onto the membrane for hybridization. A total of 112 differentially expressed sequence tags (ESTs) had been sequenced. For the host responses, 20% of ESTs (13 ESTs, 10 genes, and 1 non-coding RNA) from the forward library and 93.6% of ESTs (44 ESTs, 28 genes) from the reverse library were identified as differentially expressed genes (DEGs) of the hosts. A high percentage of DEGs involved in catalytic activity and metabolic processes revealed that the host gene expression change after N. ceranae infection might lead to an unbalance of physiological mechanism. Among the ESTs from the forward library, 75.4% ESTs (49 ESTs belonged to 24 genes) were identified as N. ceranae genes. Out of 24 N. ceranae genes, nine DEGs were subject to real-time quantitative reverse transcription PCR (real-time qRT-PCR) for validation. The results indicated that these genes were highly expressed during N. ceranae infection. Among nine N. ceranae genes, one N. ceranae gene (AAJ76_1600052943) showed the highest expression level after infection. These identified differentially expressed genes from this SSH could provide information about the pathological effects of N. ceranae. Validation of nine up-regulated N. ceranae genes reveal high potential for the detection of early nosemosis in the field and provide insight for further applications.

The microsporidia Nosema ceranae is an obligate intracellular parasite that causes honey bee mortality and contributes to colony collapse. Fumagillin is presently the only pharmacological control for N. ceranae infections in honey bees. Resistance is already emerging, and alternative controls are critically needed. Nosema spp. exhibit increased sensitivity to heat shock, a common proteotoxic stress. Thus, we hypothesized that targeting the Nosema proteasome, the major protease removing misfolded proteins, might be effective against N. ceranae infections in honey bees. Nosema genome analysis and molecular modeling revealed an unexpectedly compact proteasome apparently lacking multiple canonical subunits, but with highly conserved proteolytic active sites expected to be receptive to FDA-approved proteasome inhibitors. Indeed, N. ceranae were strikingly sensitive to pharmacological disruption of proteasome function at doses that were well tolerated by honey bees. Thus, proteasome inhibition is a novel candidate treatment strategy for microsporidia infection in honey bees.

Nosema ceranae infections in honey bees (Apis mellifera) pose a severe threat to colony health. Beekeepers have used dicyclohexylammonium fumagillin to control Nosema apis, although it may be ineffective against N. ceranae. We investigated the ability of various propolis extracts collected from Upstate New York (United States) to decrease in vivo N. ceranae infection levels when fed ad libitum to N. ceranae-infected honey bees. Propolis extracts, most notably a dichloromethane extract, significantly lowered spore levels in a dose-dependent fashion 4 days post inoculation. When testing the in vitro anti-Nosema activity of propolis extracts, we report for the first time that spore viability was unaffected after a 24 h exposure to propolis extracts. These results present evidence that propolis extracts may effectively lower Microsporidia infections in honey bees, and that direct exposure of environmental spores to propolis alone does not kill N. ceranae.

Two microsporidian species, Nosema apis and Nosema ceranae, infect honey bees (Apis mellifera) worldwide. They are obligate intracellular parasites that multiply in the epithelial lining of the bee’s midgut and cause nosemosis. N. ceranae infections were primarily found in Apis cerana and raised interest in the last decade with the discovery of their presence in the European honey bee (Apis mellifera). Nosema spp. utilizes hosts’ energetic reserves for the purpose of propagation and disrupts the digestive processes of the bee. Nosemosis reduces the lifespan of a single bee and affects the performance of the colony. It also has an economic impact through the reduction in the honey and pollen yield of severely infected colonies or even causes them to collapse. Lack of effective therapy for nosemosis is of special concern and calls for scientific attention. Although N. ceranae and N. apis are similar in many aspects, there are important differences between them such as clinical signs of infection or the ability to resist low temperatures.

Honey bees (Apis mellifera) reared in brood combs containing high levels of pesticide residues exhibit increased susceptibility to Nosema (Microsporidia) infection

The infection of honey bees, Apis mellifera L. (Hymenoptera: Apidae), by the microsporidian Nosema ceranae is one of the factors related to the increase in colony losses and the decrease in honey production observed in recent years. However, these effects seem to differ depending on the climate zone. The range and prevalence of N. ceranae have increased significantly in the last decades, with different consequences in northern and southern temperate areas. The existence of various isolates of N. ceranae from distant geographical areas, which probably exhibit different degrees of virulence, could explain the different responses of the bee to the infection. The aim of this work was to compare the effects of two N. ceranae isolates from different host populations from Argentina on honey bee survival at two ages post‐eclosion. Using cage experiments, we compared the development of infection of worker bees through the estimation of daily bee mortality and spore counts. Host subspecies identity analysis showed a strong similarity with Apis mellifera scutellata morphotype for the northern region, with a greater hybridization between subspecies with European origin toward the central and southern regions. Genetic characterization of isolates from the three regions indicated only the presence of N. ceranae. Infected bees survived longer than control bees, and bees infected at 5 days had a lower survival than those infected at 72 h with isolates from the three regions. These differences in survival matched the development of the N. ceranae infection, with differences in spore loads for infected bees at 5 days. Our studies showed that Nosema infection and survival varied among the different ages post emergence of workers, and both increased as the honey bee aged. These differences in susceptibility to infection could be related to the immune response of bees of different ages or to changes in the composition and succession of the intestinal microbiota throughout its ontogeny.

Antifungal activity of the essential oil obtained from Cryptocarya alba against infection in honey bees by Nosema ceranae.

Honey bees play an important role in food production (honey, pollen etc.), and their pollinating activity is not only essential to maintain world agriculture production but also to ensure biodiversity in different ecosystems. Nosema ceranae is a highly prevalent worldwide pathogen for honey bees that has been related to colony losses. A commercial formulation that contains fumagillin dicyclohexylamine, Fumidil B®, can control N. ceranae infection. However, the effectiveness of Fumidil B® is affected by several factors, such as storage, treatment preparation, the quantity consumed by bees etc. Indeed, UV exposure (e.g. sunlight) drastically reduces the initial concentration of fumagillin within a few hours, while temperature affects its degradation. Although laboratory tests suggest that a semisolid mixture of honey and powdered sugar is the best option to apply fumagillin, its application in syrup (250 mL per dosage) is more effective for the treatment of infected colonies. The total amount of syrup containing fumagillin ingested by honey bees is a key factor in its efficacy, and it has been found that medicated patties were not fully consumed in field trials. In honey bee colonies, the dose of 120 mg/honey bee colony at the recommended posology is effective against depopulation and colony death due to N. ceranae after 1 year, without residues being detected in honey, although reinfection could be detected 4 months after treatment ended.

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.

Alternatives to the antibiotic fumagillin for the control of Nosema ceranae, a gut parasite of the honey bee, are needed. The prebiotics eugenol, chitosan, and naringenin and the probiotic Protexin® (Enterococcus faecium) provided in sugar syrup or protein patty either in spring or fall were evaluated for their effects on N. ceranae infection, colony population, honey yield and winter survivorship using field colonies. In the first year, spring treatments with eugenol, naringenin, and Protexin® significantly reduced N. ceranae infection and increased honey production, while Protexin® also increased adult bee populations and chitosan was ineffective. Fall treatments increased survivorship and decreased N. ceranae infection the following spring. In the second year, selected compounds were further tested with a larger number of colonies per treatment and only protein patty used in the spring and sugar syrup in the fall. Protexin® and naringenin significantly decreased N. ceranae infections and increased the population of adult bees after spring treatment, but did not affect honey yields. There were no differences between treatments for colony winter mortality, but surviving colonies that had been treated with Protexin® and naringenin were significantly more populated and had lower N. ceranae spore counts than control, non-treated colonies. Protexin® and naringenin were the most promising candidates for controlling N. ceranae and promoting honey bee populations, warranting further investigation. Future research should investigate the optimal colony dose and treatment frequency to maximize colony health.

Multiple infections are common in honey bees, Apis mellifera, but the possible role of nutrition in this regard is poorly understood. Microsporidian infections, which are promoted by protein-fed, can negatively correlate with virus infections, but the role of protein nutrition for the microsporidian-virus interface is unknown. Here, we challenged naturally deformed wing virus - B (DWV-B) infected adult honey bee workers fed with or without pollen ( = protein) in hoarding cages, with the microsporidian Nosema ceranae. Bee mortality was recorded for 14 days and N. ceranae spore loads and DWV-B titers were quantified. Amongst the groups inoculated with N. ceranae, more spores were counted in protein-fed bees. However, N. ceranae infected bees without protein-diet had reduced longevity compared to all other groups. N. ceranae infection had no effect on protein-fed bee’s longevity, whereas bees supplied only with sugar-water showed reduced survival. Our data also support that protein-feeding can have a significant negative impact on virus infections in insects. The negative correlation between N. ceranae spore loads and DWV-B titers was stronger expressed in protein-fed hosts. Proteins not only enhance survival of infected hosts, but also significantly shape the microsporidian-virus interface, probably due to increased spore production and enhanced host immunity.

Nosema ceranae is a microsporidian parasite that infects the midgut epithelial cells of honey bees (Apis mellifera), causing digestive dysfunction, reduced lifespan, impaired foraging activity and flight performance, and ultimately leading to declines in colony population and productivity. The emergence of resistance to conventional treatments such as fumagillin highlights the need for sustainable and effective alternative control strategies. This study aimed to evaluate the effects of beta-glucan supplementation on food consumption, survival rate, spore load, and antioxidant status in N. ceranae-infected honey bees. A total of 1000 newly emerged, disease-free worker bees (0–3 days old) were randomly allocated into five experimental groups, each comprising four replicates with 50 bees. All bees were provided with a 50% (w/v) sucrose solution ad libitum. The experimental groups were designated as follows: Control (uninfected), N (Nosema-infected,105 spores/bee), BG0.5 (0.5% beta-glucan, uninfected), N + BG0.5 (Nosema-infected + 0.5% beta-glucan), and N + BG1 (Nosema-infected + 1% beta-glucan). Beta-glucan supplementation resulted in a marked reduction in spore load, with a statistically significant decrease observed on day 15 compared to the Nosema-infected group without supplementation (p < 0.001). A non-significant reduction in food consumption was also recorded in the N + BG groups. Malondialdehyde (MDA) levels decreased in a dose-dependent manner with increasing beta-glucan supplementation (p < 0.05). Furthermore, both beta-glucan concentrations (0.5 and 1%) significantly elevated glutathione (GSH) activity, glutathione peroxidase (GSH-Px), and catalase (CAT) levels (p < 0.05), with the 1% beta-glucan dose producing the greatest improvement in these antioxidant markers. In conclusion, beta-glucan supplementation effectively reduced N. ceranae spore load, improved survival and food consumption, alleviated oxidative stress as indicated by lower MDA levels, and enhanced antioxidant enzyme activities in infected honey bees. These results indicate that beta-glucan represents a promising dietary intervention to mitigate the adverse effects of Nosema infection.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful technology used to investigate the spatio-temporal distribution of a huge number of molecules throughout a body/tissue section. In this paper, we report the use of MALDI IMS to follow the molecular impact of an experimental infection of Apis mellifera with the microsporidia Nosema ceranae. We performed representative molecular mass fingerprints of selected tissues obtained by dissection. This was followed by MALDI IMS workflows optimization including specimen embedding and positioning as well as washing and matrix application. We recorded the local distribution of peptides/proteins within different tissues from experimentally infected versus non infected honeybees. As expected, a distinction in these molecular profiles between the two conditions was recorded from different anatomical sections of the gut tissue. More importantly, we observed differences in the molecular profiles in the brain, thoracic ganglia, hypopharyngeal glands, and hemolymph. We introduced MALDI IMS as an effective approach to monitor the impact of N. ceranae infection on A. mellifera. This opens perspectives for the discovery of molecular changes in peptides/proteins markers that could contribute to a better understanding of the impact of stressors and toxicity on different tissues of a bee in a single experiment.

Nosema ceranae (N. ceranae) infection is prevalent globally, causing a decline in bee populations and significant economic losses to apiarists. Although several methods have been proposed for diagnosing Nosema infections, limitations in these methods have hindered their broad applications. Therefore, this current study aimed to develop a specialized method for diagnosing Nosema infections. To achieve this, a sandwich enzyme-linked immunosorbent assay (ELISA) and immunochromatography assay (ICG) were developed, and their effectiveness in screening and diagnosing Nosema infection was assessed. In sandwich ELISA, the combination of the monoclonal antibodies (mAb) 19B2 and biotinylated-19B2 exhibited stronger binding affinity to the antigen than did other combinations of mAbs that were tested. Furthermore, the antigen detection limit achieved with the sandwich ELISA surpassed that previously reported with Western blotting. The ICG was designed using the same antibody combination as that used in sandwich ELISA; however, the assay exhibited a lower diagnostic ability for Nosema infection than the ELISA. The diagnostic models developed in this study offer practical applications for conducting rapid nosemosis detection tests. These innovative techniques will help to improve the timely identification and management of nosemosis.