Outcome of colonization of Apis mellifera by Nosema ceranae

A multiplex PCR-based method, in which two small-subunit rRNA regions are simultaneously amplified in a single reaction, was designed for parallel detection of honeybee microsporidians (Nosema apis and Nosema ceranae). Each of two pairs of primers exclusively amplified the 16S rRNA targeted gene of a specific microsporidian. The multiplex PCR assay was useful for specific detection of the two species of microsporidians related to bee nosemosis, not only in purified spores but also in honeybee homogenates and in naturally infected bees. The multiplex PCR assay was also able to detect coinfections by the two species. Screening of bee samples from Spain, Switzerland, France, and Germany using the PCR technique revealed a greater presence of N. ceranae than of N. apis in Europe, although both species are widely distributed. From the year 2000 onward, statistically significant differences have been found in the proportions of Nosema spp. spore-positive samples collected between and within years. In the first period examined (1999 to 2002), the smallest number of samples diagnosed as Nosema positive was found during the summer months, showing clear seasonality in the diagnosis, which is characteristic of N. apis. From 2003 onward a change in the tendency resulted in an increase in Nosema-positive samples in all months until 2005, when a total absence of seasonality was detected. A significant causative association between the presence of N. ceranae and hive depopulation clearly indicates that the colonization of Apis mellifera by N. ceranae is related to bee losses.     

Outcome of Colonization of Apis mellifera by Nosema ceranae

A multiplex PCR-based method, in which two small-subunit rRNA regions are simultaneously amplified in a single reaction, was designed for parallel detection of honeybee microsporidians (Nosema apis and Nosema ceranae). Each of two pairs of primers exclusively amplified the 16S rRNA targeted gene of a specific microsporidian. The multiplex PCR assay was useful for specific detection of the two species of microsporidians related to bee nosemosis, not only in purified spores but also in honeybee homogenates and in naturally infected bees. The multiplex PCR assay was also able to detect coinfections by the two species. Screening of bee samples from Spain, Switzerland, France, and Germany using the PCR technique revealed a greater presence of N. ceranae than of N. apis in Europe, although both species are widely distributed. From the year 2000 onward, statistically significant differences have been found in the proportions of Nosema spp. spore-positive samples collected between and within years. In the first period examined (1999 to 2002), the smallest number of samples diagnosed as Nosema positive was found during the summer months, showing clear seasonality in the diagnosis, which is characteristic of N. apis. From 2003 onward a change in the tendency resulted in an increase in Nosema-positive samples in all months until 2005, when a total absence of seasonality was detected. A significant causative association between the presence of N. ceranae and hive depopulation clearly indicates that the colonization of Apis mellifera by N. ceranae is related to bee losses.     

Nosema ceranae in European honey bees (Apis mellifera)

Nosema ceranae is a microsporidian parasite described from the Asian honey bee, Apis cerana. The parasite is cross-infective with the European honey bee, Apis mellifera. It is not known when or where N. ceranae first infected European bees, but N. ceranae has probably been infecting European bees for at least two decades. N. ceranae appears to be replacing Nosema apis, at least in some populations of European honey bees. This replacement is an enigma because the spores of the new parasite are less durable than those of N. apis. Virulence data at both the individual bee and at the colony level are conflicting possibly because the impact of this parasite differs in different environments. The recent advancements in N. ceranae genetics, with a draft assembly of the N. ceranae genome available, are discussed and the need for increased research on the impacts of this parasite on European honey bees is emphasized.     

Nosema ceranae in drone honey bees (Apis mellifera)

Nosema ceranae is a microsporidian intracellular parasite of honey bees, Apis mellifera. Previously Nosema apis was thought to be the only cause of nosemosis, but it has recently been proposed that N. ceranae is displacing N. apis. The rapid spread of N. ceranae could be due to additional transmission mechanisms, as well as higher infectivity. We analyzed drones for N. ceranae infections using duplex qPCR with species specific primers and probes. We found that both immature and mature drones are infected with N. ceranae at low levels. This is the first report detecting N. ceranae in immature bees. Our data suggest that because drones are known to drift from their parent hives to other hives, they could provide a means for disease spread within and between apiaries.     

Low natural levels of Nosema ceranae in Apis mellifera queens

Queens are the primary female reproductive individuals in honey bee colonies and, while they are generally free from Nosema ceranae infection, they are nevertheless susceptible. We sought to determine whether queens are naturally infected by N. ceranae, as these infections could be a factor in the rapid spread of this parasite. Queens were analyzed using real-time PCR and included larval queens, newly emerged, and older mated queens. Overall, we found that all tissues we examined were infected with N. ceranae at low levels but no samples were infected with Nosema apis. The infection of the ovaries and spermatheca suggests the possibility of vertical transmission of N. ceranae.     

Distribution of Nosema ceranae in the European honeybee, Apis mellifera in Japan

The microsporidian species, Nosema apis and Nosema ceranae are both known to infect the European honeybee, Apis mellifera. Nosema disease has a global distribution and is responsible for considerable economic losses among apiculturists. In this study, 336 honeybee samples from 18 different prefectures in Japan were examined for the presence of N. apis and N. ceranae using a PCR technique. Although N. ceranae was not detected in most of the apiaries surveyed, the parasite was detected at three of the sites examined. Further, N. ceranae appears to be patchily distributed across Japan and no apparent geographic difference was observed among the areas surveyed. In addition, the apparent absence of N. apis suggests that N. ceranae may be displacing N. apis in A. mellifera in Japan. Partial SSU rRNA gene sequence analysis revealed the possible existence of two N. ceranae groups from different geographic regions in Japan. It seems likely that these microsporidian parasites were introduced into Japan through the importation of either contaminated honeybee-related products or infected queens. This study confirmed that PCR detection is effective for indicating the presence of this pathogen in seemingly healthy colonies. It is therefore hoped that the results presented here will improve our understanding of the epidemiology of Nosema disease so that effective controls can be implemented.     

Experimental infection of Apis mellifera honeybees with Nosema ceranae (Microsporidia)

In this report, an experimental infection of Apis mellifera by Nosema ceranae, a newly reported microsporidian in this host is described. Nosema free honeybees were inoculated with 125,000 N. ceranae spores, isolated from heavily infected bees. The parasite species was identified by amplification and sequencing the SSUrRNA gene of the administered spores. Three replicate cages of 20 honeybees each were prepared, along with one control cage (n=20) supplied with sugar syrup only. The infection rate was 100% at the dosage administered. The presence of Nosema inside ventricular cells was confirmed in the samples using ultrathin sectioning and transmission electron microscopy. By day 3 p.i. a few cells (4.4%+/-1.2) were observed to be parasitized, whereas by 6 days p.i. more than half of the counted cells (66.4%+/-6) showed different parasite stages, this value increasing on day 7 p.i. (81.5%+/-14.8). Only one control bee died on day 7 p.i. In the infected groups, mortality was not observed until day 6 p.i. (66.7%+/-5.6). Total mortality on day 7 p.i. was 94.1% in the three infected replicates and by day 8 p.i. no infected bee was alive. After the infection, the parasites invaded both the tip of folds and the basal cells of the epithelium and the autoinfective capacity of the spores seemed to spread the infection rapidly between epithelial cells. On day 3 p.i., mature spores could be seen inside host cell tissue implying that the developmental cycle had been completed. The large number of parasitized cells, even the regenerative ones, the presence of autoinfective spores and the high mortality rate demonstrate that N. ceranae is highly pathogenic to Apis mellifera. Possible relation with bee depopulation syndrome is discussed by authors.     

Pathological effects of the microsporidium Nosema ceranae on honey bee queen physiology (Apis mellifera)

Nosema ceranae, a microsporidian parasite originally described in the Asian honey bee Apis cerana, has recently been found to be cross-infective and to also parasitize the European honey bee Apis mellifera. Since this discovery, many studies have attempted to characterize the impact of this parasite in A. mellifera honey bees. Nosema species can infect all colony members, workers, drones and queens, but the pathological effects of this microsporidium has been mainly investigated in workers, despite the prime importance of the queen, who monopolizes the reproduction and regulates the cohesion of the society via pheromones. We therefore analyzed the impact of N. ceranae on queen physiology. We found that infection by N. ceranae did not affect the fat body content (an indicator of energy stores) but did alter the vitellogenin titer (an indicator of fertility and longevity), the total antioxidant capacity and the queen mandibular pheromones, which surprisingly were all significantly increased in Nosema-infected queens. Thus, such physiological changes may impact queen health, leading to changes in pheromone production, that could explain Nosema-induced supersedure (queen replacement).     

First detection of Nosema ceranae, a microsporidian parasite of European honey bees (Apis mellifera), in Canada and central USA

Nosema ceranae is an emerging microsporidian parasite of European honey bees, Apis mellifera, but its distribution is not well known. Six Nosema-positive samples (determined from light microscopy of spores) of adult worker bees from Canada (two each from Nova Scotia, New Brunswick, and Prince Edward Island) and two from USA (Minnesota) were tested to determine Nosema species using previously-developed PCR primers of the 16S rRNA gene. We detected for the first time N. ceranae in Canada and central USA. One haplotype of N. ceranae was identified; its virulence may differ from that of other haplotypes.     

Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera

The economically most important honey bee species, Apis mellifera, was formerly considered to be parasitized by one microsporidian, Nosema apis. Recently, [Higes, M., Martín, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93-95] and [Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A Nosema ceranae isolate from the honeybee Apis mellifera. Apidologie 38, 30-37] used 16S (SSU) rRNA gene sequences to demonstrate the presence of Nosema ceranae in A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between N. apis and N. ceranae based on PCR-RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (N =9 isolates gave N. apis RFLPs and sequences, N =20 isolates gave N. ceranae RFLPs and sequences; 100% correct classification). We then employed the method to analyze N =115 isolates from across the world. Our data, combined with N =36 additional published sequences demonstrate that (i) N. ceranae most likely jumped host to A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii) N. ceranae is now a parasite of A. mellifera across most of the world. The rapid, long-distance dispersal of N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping.     

Differential expression of immune genes of adult honey bee (Apis mellifera) after inoculated by Nosema ceranae

Nosema ceranae is a microsporidium parasite infecting adult honey bees (Apis mellifera) and is known to affects at both the individual and colony level. In this study, the expression levels were measured for four antimicrobial peptide encoding genes that are associated with bee humoral immunity (defensin, abaecin, apidaecin, and hymenoptaecin), eater gene which is a transmembrane protein involved cellular immunity and gene encoding female-specific protein (vitellogenin) in honey bees when inoculated by N. ceranae. The results showed that four of these genes, defensin, abaecin, apidaecin and hymenoptaecin were significantly down-regulated 3 and 6days after inoculations. Additionally, antimicrobial peptide expressions did not significantly differ between control and inoculated bees after 12days post inoculation. Moreover, our results revealed that the mRNA levels of eater and vitellogenin did not differ significantly following N. ceranae inoculation. Therefore, in this study we reaffirmed that N. ceranae infection induces host immunosuppression.     

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

Honey bee samples collected between 1995 and 2007 from 12 states were examined for the presence of Nosema infections. Our results showed that Nosema ceranae is a wide-spread infection of the European honey bee, Apis mellifera in the United States. The discovery of N. ceranae in bees collected a decade ago indicates that N. ceranae was transferred from its original host, Apis cerana to A. mellifera earlier than previously recognized. The spread of N. ceranae infection in A. mellifera warrants further epidemiological studies to identify conditions that resulted in such a widespread infection.     

Morphological,Molecular, and Phylogenetic Characterization of Nosema ceranae,a Microsporidian Parasite Isolated from the European Honey Bee,Apis mellifera1

ABSTRACT. Nosema ceranae, a microsporidian parasite originally described from Apis cerana, has been found to infect Apis melllifera and is highly pathogenic to its new host. In the present study, data on the ultrastructure of N. ceranae, presence of N. ceranae-specific nucleic acid in host tissues, and phylogenetic relationships with other microsporidia species are described. The ultrastructural features indicate that N. ceranae possesses all of the characteristics of the genus Nosema. Spores of N. ceranae measured approximately 4.4 × 2.2 μm on fresh smears. The number of coils of the polar filament inside spores was 18–21. Polymerase chain reaction (PCR) signals specific for N. ceranae were detected not only in the primary infection site, the midgut, but also in the tissues of hypopharyngeal glands, salivary glands, Malpighian tubules, and fat body. The detection rate and intensity of PCR signals in the fat body were relatively low compared with other examined tissues. Maximum parsimony analysis of the small subunit rRNA gene sequences showed that N. ceranae appeared to be more closely related to the wasp parasite, Nosema vespula, than to N. apis, a parasite infecting the same host.     

Biodiversity of Apis mellifera populations from Tenerife (Canary Islands) and hybridisation with East European races

The biodiversity of honeybee (Apis mellifera) populations from Tenerife (Canary Islands, Spain) has been assessed by restriction analysis of a mitochondrial non-coding intergenic region. Seventy-nine colonies were analysed from thirteen apiaries in six populations that have been kept from recent queen introduction. The length and restriction pattern of the PCR amplified products of the intergenic region identified four mitochondrial haplotypes. One of these haplotypes shows the same restriction pattern and composition of the intergenic region carried by honeybees belonging to the African lineage. Two haplotypes are characterised by a particular intergenic region found with high frequency in the Canarian populations. The haplotype representative of the East European honeybee lineage shows a frequency of 35%, thus indicating introduction of queen honeybees. The finding of this haplotype in Canarian honeybees suggests that hybridisation between the endemic Apis mellifera populations and imported bees is occurring in Tenerife.     

Protective potential of Chinese herbal extracts against microsporidian Nosema ceranae,an emergent pathogen of western honey bees,Apis mellifera L.

Nosema ceranae, a newly emergent parasite invading western honey bees (Apis mellifera L.), is indicated to threaten honey bee health at both individual and colony levels. However, the efficient and environmentally-friendly treatments are quite limited at present. To find alternative medicine to control Nosema diseases, the effect of 8 types of herbal extracts against N. ceranae infection were screened under laboratory condition. Of which, 1% Andrographis paniculata (A. paniculata) decoction was found to significantly decrease N. ceranae spore numbers on 7 days post infection (dpi) and 13 dpi. Then, our results further revealed that A. paniculata decoction at doses ranging from 1% to 7% displayed significant efficient inhibition of Nosema spore proliferation and improved the infected bees' survival rates in a dose-dependent manner. A. paniculata decoction was found to protect the gut tissues of infected workers from damage cause by N. ceranae, which might be due to the regulation of the expression of certain genes in Wnt and JNK pathways, including armadillo, basket, frizzled2 and groucho. Additionally, our study suggested that A. paniculata decoction performed this Nosema spore-reducing potential over its two monomers, andrographolide and dehydrographolide. Taken together, this work enables us to better understand A. paniculata decoction's potential to inhibit N. ceranae infection, thus providing a new guidance for developing applicable drugs to control Nosema diseases.     

Local honeybee (Apis mellifera mellifera L.) populations in the Urals

The COI-COII intergenic region of mitochondrial DNA (mtDNA) was studied in local honeybee (Apis mellifera mellifera) L. populations from the Middle and Southern Urals. Analysis of bee colonies in these regions revealed apiaries enriched in families descending from A. m. mellifera in the maternal lineage. These results confirm the suggestion of preservation of A. m. mellifera refuges in the Urals and provide grounds for work on the preservation of the gene pool of this bee variety, valuable for all Russia.     

The levels of natural Nosema spp. infection in Apis mellifera iberiensis brood stages

Nosema ceranae is the most prevalent endoparasite of Apis mellifera iberiensis and it is a major health problem for bees worldwide. The infective capacity of N. ceranae has been demonstrated experimentally in honey bee brood, however no data are available about its prevalence in brood under natural conditions. Thus, brood combs from 10 different hives were analyzed over two consecutive years, taking samples before and after winter. A total of 1433 larvae/pupae were analyzed individually and N. ceranae (3.53%) was the microsporidian most frequently detected, as opposed to Nosema apis (0.42%) which was more frequently detected in conjunction with N. ceranae (0.71%). The active multiplication of both microsporidians was confirmed by the expression (real-time-PCR) of the N. ceranae polar tube protein 3 gene and/or the N. apis RNA polymerase II gene in 24% of the brood samples positive for Nosema spp. Both genes are related to microsporidian multiplication. As such, N. ceranae multiplication was confirmed in 1.06% of the samples, while N. apis multiplication was only observed in co-infections with N. ceranae (0.07%). Brood cells were analyzed for the presence of Nosema spp., as those are the immediate environment where the brood stages develop. The brood samples infected by Nosema spp. were in brood cells in which that microsporidians were not detected, while brood cells positive for N. ceranae hosted brood stages that were not apparently infected, indicating that this is unlikely to be the main pathway of infection. Finally, the colonies with brood infected by N. ceranae showed higher levels (numbers) of infected adult bees, although the differences were not significant before (P = 0.260), during (P = 0.055) or after (P = 0.056) brood sampling. These results show that N. ceranae is a bee parasite ubiquitous to all members of the colony, irrespective of the age of the bee. It is also of veterinary interest and should be considered when studying the epidemiology of the disease.     

Ultrastructure of the midgut of the worker honey bee Apis mellifera heavily infected with Nosema apis

Midgut epithelial cells from healthy bees possessed numerous mitochondria, strands of endoplasmic reticulum, evenly distributed ribosomes, zymogen granules, and two kinds of lipid inclusions. In heavily infected midguts of honey bees, Apis mellifera, all epithelial cells were observed to be infected with Nosema apis. Cells of the entire midgut were packed with mature spores and, in some cases, mixed with immature stages. Spores were not found among cells of the brush border and basal infolding. Muscle cells and tracheal end cells of the midgut were not infected. The cytoplasm of the infected cell contained a large number of vacuoles, numerous large inclusion bodies, and aggregated ribosomes. Signs of extensive lysis were observed within the heavily infected cells, although the cell membranes were intact.