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.     

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

Nosema ceranae and pesticide exposure can contribute to honey bee health decline. Bees reared from brood comb containing high or low levels of pesticide residues were placed in two common colony environments. One colony was inoculated weekly with N. ceranae spores in sugar syrup and the other colony received sugar syrup only. Worker honey bees were sampled weekly from the treatment and control colonies and analyzed for Nosema spore levels. Regardless of the colony environment (spores+syrup added or syrup only added), a higher proportion of bees reared from the high pesticide residue brood comb became infected with N. ceranae, and at a younger age, compared to those reared in low residue brood combs. These data suggest that developmental exposure to pesticides in brood comb increases the susceptibility of bees to N. ceranae infection.     

How natural infection by Nosema ceranae causes honeybee colony collapse

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.     

Survival and immune response of drones of a Nosemosis tolerant honey bee strain towards N. ceranae infections

Honey bee colonies (Apis mellifera) have been selected for low level of Nosema in Denmark over decades and Nosema is now rarely found in bee colonies from these breeding lines. We compared the immune response of a selected and an unselected honey bee lineage, taking advantage of the haploid males to study its potential impact on the tolerance toward Nosema ceranae, a novel introduced microsporidian pathogen. After artificial infections of the N. ceranae spores, the lineage selected for Nosema tolerance showed a higher N. ceranae spore load, a lower mortality and an up-regulated immune response. The differences in the response of the innate immune system between the selected and unselected lineage were strongest at day six post infection. In particular genes of the Toll pathway were up-regulated in the selected strain, probably is the main immune pathway involved in N. ceranae infection response. After decades of selective breeding for Nosema tolerance in the Danish strain, it appears these bees are tolerant to N. ceranae infections.     

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, a new microsporidian parasite in honeybees in Europe

Twelve samples of adult honey bees from different regions of Spain from colonies with clear signs of population depletion, positive to microsporidian spores using light microscopy (1% of total positive samples analysed), were selected for molecular diagnosis. PCR specific primers for a region of the 16S rRNA gene of Microsporidia were developed and the PCR products were sequenced and compared to GenBank entries. The sequenced products of 11 out of the 12 samples were identical to the corresponding Nosema ceranae sequence. This is the first report of N. ceranae in colonies of Apis mellifera in Europe. The suggested link of the infections to clinical disease symptoms makes imperative a study of the virulence of N. ceranae in European races of honey bees.     

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.     

Immune suppression in the honey bee (Apis mellifera) following infection by Nosema ceranae (Microsporidia)

Two microsporidia species have been shown to infect Apis mellifera , Nosema apis and Nosema ceranae . This work present evidence that N. ceranae infection significantly suppresses the honey bee immune response, although this effect was not observed following infection with N. apis . Immune suppression would also increase susceptibility to other bee pathogens and senescence. Despite the importance of both Nosema species in honey bee health, there is no information about their effect on the bees' immune system and present results can explain the different virulence between both microsporida infecting honeybees.     

Asymptomatic presence of Nosema spp. in Spanish commercial apiaries

Nosemosis is caused by intracellular parasites (Nosema apis and Nosema ceranae) that infect the midgut epithelial cells in adult honey bees. Recent studies relate N. ceranae to Colony Collapse Disorder and there is some suggestion that Nosema spp., especially N. ceranae, induces high mortality in honey bees, a fact that is considered as a serious threat for colony survival. 604 samples of adult honey bees for Nosema spp. analysis were collected from beekeeping colonies across Spain and were analysed using PCR with capillary electrophoresis. We also monitored 77 Andalusian apiaries for 2years; the sampled hives were standard healthy colonies, without any special disease symptoms. We found 100% presence of Nosema spp. in some locations, indicating that this parasite was widespread throughout the country. The two year monitoring indicated that 87% of the hives with Nosema spp. remained viable, with normal honey production and biological development during this period of time. The results of these trials indicated that both N. ceranae and N. apis could be present in these beehives without causing disease symptom and that there is no evidence for the replacement of N. apis by N. ceranae, supporting the hypothesis that nosemosis is not the main reason of the collapse and death of beehives.     

Varroa destructor infestation in untreated honey bee (Hymenoptera: Apidae) colonies selected for hygienic behavior

Honey bee (Apis mellifera L.) colonies bred for hygienic behavior were tested in a large field trial to determine if they were able to resist the parasitic mite Varroa destructor better than unselected colonies of"Starline" stock. Colonies bred for hygienic behavior are able to detect, uncap, and remove experimentally infested brood from the nest, although the extent to which the behavior actually reduces the overall mite-load in untreated, naturally infested colonies needed further verification. The results indicate that hygienic colonies with queens mated naturally to unselected drones had significantly fewer mites on adult bees and within worker brood cells than Starline colonies for up to 1 yr without treatment in a commercial, migratory beekeeping operation. Hygienic colonies actively defended themselves against the mites when mite levels were relatively low. At high mite infestations (>15% of worker brood and of adult bees), the majority of hygienic colonies required treatment to prevent collapse. Overall, the hygienic colonies had similar adult populations and brood areas, produced as much honey, and had less brood disease than the Starline colonies. Thus, honey bees bred for hygienic behavior performed as well if not better than other commercial lines of bees and maintained lower mite loads for up to one year without treatment.     

Comparative analysis of thermoregulation in the nests of certain species of social insects

Studies have been made on thermal regulation in the nests of families of the honey bee Apis mellifera, wasp Dolihovespula silvestris and bumblebees Bombus terrestris, B. agrorum and B. lapidaris during their maximum development. It was shown that thermoregulation significantly stimulated the brood in the nest. Among the species investigated, the highest thermoregulatory capacities are exhibited by honey bees, this fact being associated with the large number of individuals in their colonies. During cooling, bees group around the brood and their bodies make a cover of thermal insulation. Sheltering of the nests plays the main role in heat preservation of wasps and bumblebees. The latter, using contact method of heating of cells with the brood, provide rather constant temperature for its development. Honey bees, wasps and bumblebees react to overheating in the nests essentially in a similar way, i. e. by active aeration of their nests by vigorous wing beatings. The frequency of beatings increases with the increase in temperature.     

Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)

Through the use of proboscis-extension reflex conditioning, we demonstrate that honey bees (Apis mellifera L.) bred for hygienic behavior (a behavioral mechanism of disease resistance) are able to discriminate between odors of healthy and diseased brood at a lower stimulus level than bees from a non-hygienic line. Electroantennogram recordings confirmed that hygienic bees exhibit increased olfactory sensitivity to low concentrations of the odor of chalkbrood infected pupae (a fungal disease caused by Ascosphaera apis). Three-week-old hygienic bees were able to discriminate between the brood odors significantly better than three-week old non-hygienic bees. However, the differential performance in brood odor discrimination was primarily genetically based, not a direct result of age, experience, or the temporary behavioral state of the bee. Lower stimulus thresholds for both the olfactory and behavioral responses of hygienic bees may facilitate their ability to detect, uncap and remove diseased brood rapidly from the nest. In contrast, non-hygienic bees, possessing higher response thresholds, may not be able to detect diseased brood as easily. Our results provide an example of how physiological and behavioral differences between the hygienic and non-hygienic honey bee lines, operating at the level of the individual, could produce colony-specific behavioral phenotypes.     

High genetic variability of Nosema ceranae populations in Apis mellifera from East Asia compared to central Asia and the Americas

Biological Invasions - Nosema ceranae is believed to have been originally a parasite of the Asian honey bee, Apis cerana, in East Asia that later infected the Western honey bee, Apis mellifera,...     

Is the number of antennal plate organs (sensilla placodea) greater in hygienic than in non-hygienic Africanized honey bees?

Hygienic behavior is a desirable trait in honey bees (Apis mellifera L.), as hygienic bees quickly remove diseased brood, interrupting the infectious cycle. Hygienic lines of honey bees appear to be more sensitive to the odors of dead and diseased honey bee brood, and Africanized honey bees are generally more hygienic than are European honey bees. We compared the number of sensilla placodea, antennal sensory structures involved in the perception of odor, in 10 bees from each of six hygienic and four non-hygienic colonies of Africanized honey bees. The sensilla placodea of three of the terminal segments (flagellomeres) of the right antenna of each bee were counted with a scanning electron microscope. There were no significant differences in the mean numbers of sensilla placodea between the hygienic and non-hygienic bees, though the variance was higher in the hygienic group. Flagellomere 4 had significantly more sensilla placodea than flagellomeres 6 and 8. However, there was no significant difference between the other two flagellomeres. As hygienic bees are capable of identifying dead, injured, or infested brood inside a capped brood cell, sensilla placodea probably have an important role in enabling worker bees to sense sick brood. However, we did not find greater numbers of this sensory structure in the antennae of hygienic, compared to non-hygienic Africanized honey bees.     

Nosema ceranae in age cohorts of the western honey bee (Apis mellifera)

Nosemaceranae intensity (mean spores per bee) and prevalence (proportion of bees infected in a sample) were analyzed in honey bees of known ages. Sealed brood combs from five colonies were removed, emerging bees were marked with paint, released back into their colonies of origin, and collected as recently emerged (0-3 days old), as house bees (8-11 days old), and as foragers (22-25 days old). Fifty bees from each of the five colonies were processed individually at each collection date for the intensity and prevalence of N. ceranae infection. Using PCR and specific primers to differentiate Nosema species, N. ceranae was found to be the only species present during the experiment. At each collection age (recent emergence, house, forager) an additional sample from the inner hive cover (background bees=BG) of each colony was collected to compare the N. ceranae results of this sampling method, commonly used for Nosema spore quantification, to the samples comprised of marked bees of known ages. No recently emerged bees exhibited infection with N. ceranae. One house bee out of the 250 individuals analyzed (prevalence=0.4%) tested positive for N. ceranae, at an infection level of 3.35×10(6) spores. Infection levels were not statistically different between the recently emerged (mean=0 spores/bee) and house bees (mean=1.34×10(4) spores/bee) (P=0.99). Foragers exhibited the highest prevalence (8.3%) and infection intensity (mean=2.38×10(6) spores/bee), with a range of 0-8.72×10(7) spores in individual bees. The average infection level across all foragers was significantly higher than that of recently emerged bees (P=0.01) and house bees (P=0.01). Finally, the prevalence of Nosema in infected bees was found to be positively correlated with the infection intensity in the sample.     

The comparison of rDNA spacer regions of Nosema ceranae isolates from different hosts and locations

Nosema ceranae is a common microsporidian pathogen, one of two Nosema species that cause "nosema disease" in honeybees, Apis cerana and Apis mellifera. Samples of N. ceranae rDNA from isolates collected in different locations were sequenced and one 5S rRNA was found to be upstream of SSUrRNA. The rDNA arrangement, 5'-5S rRNA-IGS-SSUrRNA-ITS-LSUrRNA-3', was found in all isolates. In order to better understand the distribution relationship between N. ceranae isolates from A. cerana and A. mellifera, their rRNA spacer regions were also sequenced for analysis. Results showed that there are no significant differences between the IGS sequences of the isolates and no difference in the ITS sequence with the exception of one transition found in an isolate from Martinique. These isolates showed consistency in the IGS phylogenic analysis suggesting that no transmission barrier exists between A. mellifera and A. cerana and there is no difference between isolates from geography separated areas.     

Managing honey bees (Hymenoptera: Apidae) for greenhouse tomato pollination

Although commercially reared colonies of bumble bees (Bombus sp.) are the primary pollinator world-wide for greenhouse tomatoes (Lycopersicon esculentum Mill.) previous research indicates that honey bees (Apis mellifera L.) might be a feasible alternative or supplement to bumble bee pollination. However, management methods for honey bee greenhouse tomato pollination scarcely have been explored. We 1) tested the effect of initial amounts of brood on colony population size and flight activity in screened greenhouses during the winter, and 2) compared foraging from colonies with brood used within screened and unscreened greenhouses during the summer. Brood rearing was maintained at low levels in both brood and no-brood colonies after 21 d during the winter, and emerging honey bees from both treatments had significantly lower weights than bees from outdoor colonies. Honey bee flight activity throughout the day and over the 21 d in the greenhouse was not influenced by initial brood level. In our summer experiment, brood production in screened greenhouses neared zero after 21 d but higher levels of brood were reared in unscreened greenhouses with access to outside forage. Flower visitation measured throughout the day and over the 21 d the colonies were in the greenhouse was not influenced by screening treatment. An economic analysis indicated that managing honey bees for greenhouse tomato pollination would be financially viable for both beekeepers and growers. We conclude that honey bees can be successfully managed for greenhouse tomato pollination in both screened and unscreened greenhouses if the foraging force is maintained by replacing colonies every 3 wk.     

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

Nosema ceranae is now considered to be an emerging infectious disease of the European honey bee Apis mellifera. Only one antibiotic, Fumagillin, is commercially available to combat Nosema infections. This antibiotic treatment is banned from use in Europe and elsewhere there is a high probability for antibiotic resistance to develop. We are therefore interested in investigating the effects of a natural propolis extract on its ability to reduce N. ceranae infection loads in the dwarf honey bee, Apis florea, a native honey bee with a range that overlaps with Apis cerana and Apis mellifera that is at risk of infection. Experimentally infected caged bees were fed a treatment consisting of 0%, 50%, or 70% propolis extract. All 50% and 70% propolis treated bees had significantly lower infection loads, and the 50% treated bees had higher survival in comparison to untreated bees. In addition, propolis treated bees had significantly higher haemolymph trehalose levels and hypopharyngeal gland protein content similar to levels of uninfected bees. Propolis ethanolic extract treatment could therefore be considered as a possible viable alternative to Fumagillin to improve bee health. This natural treatment deserves further exploration to develop it as a possible alternative to combat N. ceranae infections distributed around the world.