First evaluation of the Vairimorpha (Nosema) ceranae genome’s DNA replication genes as targets for RNA interference-mediated suppression of the honey bee Apis mellifera nosemosis

Chronic Nosema ceranae infection inflicts comprehensive and persistent immunosuppression and accelerated lipid loss in host Apis mellifera honey bees

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.

Nosema ceranae is a long-present and wide-spread microsporidian infection of the European honey bee ( Apis mellifera) in the United States

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.

Survival and health improvement of Nosema infected Apis florea (Hymenoptera: Apidae) bees after treatment with propolis extract

The Asian honey bee Apis cerana and the European honey bee Apis mellifera are closely related and morphologically very similar. Where these species coexist, they appear to compete, but the outcomes of competition vary enormously between locations. Here, we report comparative behavioural data for A. cerana and A. mellifera in China gathered by tracking bees using radio frequency identification. Both species organise their division of labour by temporal polyethism and have remarkably similar demographic structure. Analyses of the homing capacities of both species following large-scale displacement suggest that A. mellifera colonies have a larger range than A. cerana. We observed that relocation of A. mellifera to a new environment disrupted colony function for 3 weeks. Our data show that A. mellifera and. A cerana occupy extremely similar behavioural niches, and therefore, the potential for competition between these species is very high.

SummaryNosema apis and Nosema ceranae are causative agents of Nosemosis in the honey bee Apis mellifera, although N. ceranae may cause a more virulent disease. Selection of colonies with high tolerance to N. ceranae could be important for reducing problems caused by this pathogen. The aim of the present work was to evaluate the existence of honey bee colonies with different degrees of N. ceranae infection and test if this difference could be related to the immune response or vitellogenin expression. Healthy honey bee colonies were relocated to a plantation of Eucalyptusgrandisto favour natural infection of N. ceranae. Fifteen and thirty days after relocation, the proportion of infected bees and the number of N. ceranae spores per field were quantified. The colonies with higher and lower levels of infection (HL and LL, respectively) were selected. Newly emerged bees from both colonies were artificially infected with N. ceranaeand seven days after infection the expression of immune related genes and vitellogenin was evaluated by real time PCR. No significant differences were observed in expression of abaecin, hymenoptaecin, defensin, glucose dehydrogenase or lysozyme mRNA levels between infected bees from HL and LL colonies or between control bees from both colonies. Vitellogenin expression was higher in bees from the LL colony than in bees from the HL colony, when infected or control bees were compared between them. This protein possesses pleiotropic effects and is a central element in the life-history of honey bees. For that reason, its differential expression could be associated with resistance to N. ceranae.

Simple SummaryNosema disease of honey bees is caused by the fungus Nosema ceranae, which multiplies and damages cells lining the digestive tract, impairing food digestion and debilitating the bees. Current control involves using antibiotics, which is undesirable because of possible antibiotic resistance of the fungus and contamination of honey. In this study, the natural compounds flagellin, zymosan, chitosan and peptidoglycan were investigated as alternatives for controlling Nosema ceranae infections and for their effect on bee survivorship and behaviors. Chitosan and peptidoglycan reduced infection and increased survivorship of infected bees. However, neither compound altered the bees’ hygienic behavior, which was also not affected by the infection. Chitosan treated bees collected more pollen and nectar than healthy and infected bees. Memory in the bees was temporarily impaired by chitosan but was not affected by peptidoglycan, nor was it affected by Nosema ceranae. This study shows that chitosan and peptidoglycan provide benefits by partially reducing Nosema ceranae infection while increasing survivorship of honey bees. Also, chitosan and peptidoglycan increased the collection of pollen and nectar, which may improve bee health and colony productivity. These benefits could result in more honey produced, more crops pollinated and more healthy bee colonies.Nosema disease is a major disease of honey bees caused by two species of microsporidia, Nosema apis and N. ceranae. Current control involves using antibiotics, which is undesirable because of possible antibiotic resistance and contamination. In this study, flagellin, zymosan, chitosan, and peptidoglycan were investigated as alternatives for controlling N. ceranae infections and for their effect on bee survivorship and behaviors. Chitosan and peptidoglycan significantly reduced the infection, and significantly increased survivorship of infected bees, with chitosan being more effective. However, neither compound altered the bees’ hygienic behavior, which was also not affected by the infection. Chitosan significantly increased pollen foraging and both compounds significantly increased non-pollen foraging compared to healthy and infected bees. Memory retention, evaluated with the proboscis extension reflex assay, was temporarily impaired by chitosan but was not affected by peptidoglycan, nor was it affected by N. ceranae infection compared to the non-infected bees. This study indicates that chitosan and peptidoglycan provide benefits by partially reducing N. ceranae spore numbers while increasing survivorship compared to N. ceranae infected bees. Also, chitosan and peptidoglycan improved aspects of foraging behavior even more than in healthy bees, showing that they may act as stimulators of important honey bee behaviors.

In recent years, honeybees (Apis mellifera) have been strangely disappearing from their hives, and strong colonies have suddenly become weak and died. The precise aetiology underlying the disappearance of the bees remains a mystery. However, during the same period, Nosema ceranae, a microsporidium of the Asian bee Apis cerana, seems to have colonized A. mellifera, and it's now frequently detected all over the world in both healthy and weak honeybee colonies. For first time, we show that natural N. ceranae infection can cause the sudden collapse of bee colonies, establishing a direct correlation between N. ceranae infection and the death of honeybee colonies under field conditions. Signs of colony weakness were not evident until the queen could no longer replace the loss of the infected bees. The long asymptomatic incubation period can explain the absence of evident symptoms prior to colony collapse. Furthermore, our results demonstrate that healthy colonies near to an infected one can also become infected, and that N. ceranae infection can be controlled with a specific antibiotic, fumagillin. Moreover, the administration of 120 mg of fumagillin has proven to eliminate the infection, but it cannot avoid reinfection after 6 months. We provide Koch's postulates between N. ceranae infection and a syndrome with a long incubation period involving continuous death of adult bees, non-stop brood rearing by the bees and colony loss in winter or early spring despite the presence of sufficient remaining pollen and honey.

Nosemosis C, a Nosema disease caused by microsporidia parasite Nosema ceranae, is a significant disease burden of the European honey bee Apis mellifera which is one of the most economically important insect pollinators. Nevertheless, there is no effective treatment currently available for Nosema disease and the disease mechanisms underlying the pathological effects of N. ceranae infection in honey bees are poorly understood. Iron is an essential nutrient for growth and survival of hosts and pathogens alike. The iron tug-of-war between host and pathogen is a central battlefield at the host-pathogen interface which determines the outcome of an infection, however, has not been explored in honey bees. To fill the gap, we conducted a study to investigate the impact of N. ceranae infection on iron homeostasis in honey bees. The expression of transferrin, an iron binding and transporting protein that is one of the key players of iron homeostasis, in response to N. ceranae infection was analysed. Furthermore, the functional roles of transferrin in iron homeostasis and honey bee host immunity were characterized using an RNA interference (RNAi)-based method. The results showed that N. ceranae infection causes iron deficiency and upregulation of the A. mellifera transferrin (AmTsf) mRNA in honey bees, implying that higher expression of AmTsf allows N. ceranae to scavenge more iron from the host for its proliferation and survival. The suppressed expression levels of AmTsf via RNAi could lead to reduced N. ceranae transcription activity, alleviated iron loss, enhanced immunity, and improved survival of the infected bees. The intriguing multifunctionality of transferrin illustrated in this study is a significant contribution to the existing body of literature concerning iron homeostasis in insects. The uncovered functional role of transferrin on iron homeostasis, pathogen growth and honey bee’s ability to mount immune responses may hold the key for the development of novel strategies to treat or prevent diseases in honey bees.

Honey bee (Apis mellifera) health has been severely impacted by multiple environmental stressors including parasitic infection, pesticide exposure, and poor nutrition. The decline in bee health is therefore a complex multifactorial problem which requires a holistic investigative approach. Within the exposome paradigm, the combined exposure to the environment, drugs, food, and individuals’ internal biochemistry affects health in positive and negative ways. In the context of the exposome, honey bee hive infection with parasites such as Nosema ceranae is also a form of environmental exposure. In this study, we hypothesized that exposure to xenobiotic pesticides and other environmental chemicals increases susceptibility to N. ceranae infection upon incidental exposure to the parasite. We further queried whether these exposures could be linked to changes in conserved metabolic biological pathways. From 30 hives sampled across 10 sites, a total of 2,352 chemical features were found via gas chromatography-time of flight mass spectrometry (GC-TOF) in extracts of honey bees collected from each hive. Of these, 20 pesticides were identified and annotated, and found to be significantly associated with N. ceranae infection. We further determined that infected hives were linked to a greater number of xenobiotic exposures, and the relative concentration of the exposures were not linked to the presence of a N. ceranae infection. In the exposome profiles of the bees, we also found chemicals inherent to known biological metabolic pathways of Apis mellifera and identified 9 dysregulated pathways. These findings have led us to posit that for hives exposed to similar chemicals, those that incur multiple, simultaneous xenobiotic stressors have a greater incidence of infection with N. ceranae. Mechanistically, our results suggests the overwhelming nature of these exposures negatively affects the biological functioning of the bee, and could explain how the decline in bee populations is associated with pesticide exposures.

Honeybees (Apis mellifera) are constantly subjected to many biotic stressors including parasites. This study examined honeybees infected with Nosema ceranae (N. ceranae). N. ceranae infection increases the bees energy requirements and may contribute to their decreased survival. RNA-seq was used to investigate gene expression at days 5, 10 and 15 Post Infection (P.I) with N. ceranae. The expression levels of genes, isoforms, alternative transcription start sites (TSS) and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation suggesting that bees use a range of tactics to cope with the stress of N. ceranae infection. N. ceranae infection may cause reduced immune function in the bees by: (i)disturbing the host amino acids metabolism (ii) down-regulating expression of antimicrobial peptides (iii) down-regulation of cuticle coatings and (iv) down-regulation of odorant binding proteins.

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

Nosema ceranae is an emerging pathogen of the western honey bee (Apis mellifera L.), and thus its seasonality and impact on bee colonies is not sufficiently documented for North America. This study was conducted to determine the infection intensity, prevalence, and viability of N. ceranae in >200 honey bee colonies during spring, summer, and fall, in a North American region. We also determined the relationship of N. ceranae infections with colony populations, food stores, bee survivorship, and overwinter colony mortality. The highest rates of N. ceranae infection, prevalence, and spore viability were found in the spring and summer, while the lowest were recorded in the fall. N. ceranae spore viability was significantly correlated with its prevalence and infection intensity in bees. Threshold to high levels of N. ceranae infections (>1,000,000 spores/bee) were significantly associated with reduced bee populations and food stores in colonies. Furthermore, worker bee survivorship was significantly reduced by N. ceranae infections, although no association between N. ceranae and winter colony mortality was found. It is concluded that N. ceranae infections are highest in spring and summer and may be detrimental to honey bee populations and colony productivity. Our results support the notion that treatment is justified when infections of N. ceranae exceed 1,000,000 spores/bee.

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.