Linking evolutionary lineage with parasite and pathogen prevalence in the Iberian honey bee

The recent decline in honey bee colonies observed in both European countries and worldwide is of great interest and concern, although the underlying causes remain poorly understood. In recent years, growing evidence has implicated parasites and pathogens in this decline of both the vitality and number of honey bee colonies. The Iberian Peninsula provides an interesting environment in which to study the occurrence of pathogens and parasites in the host honey bee populations due to the presence of two evolutionary lineages in A. m. iberiensis (Western European [M] or African [A]). Here, we provide the first evidence linking the population structure of the Iberian honey bee with the prevalence of some of its most important parasites and pathogens: the Varroa destructor mite and the microsporidia Nosema apis and Nosema ceranae. Using data collected in two surveys conducted in 2006 and 2010 in 41 Spanish provinces, the evolutionary lineage and the presence of the three parasitic organisms cited above were analyzed in a total of 228 colonies. In 2006 N. apis was found in a significantly higher proportion of M lineage honey bees than in the A lineage. However, in 2010 this situation had changed significantly due to a higher prevalence of N. ceranae. We observed no significant relationships in either year between the distributions of V. destructor or N. ceranae and the evolutionary lineage present in A. m. iberiensis colonies, but the effects of these organisms on the genetic diversity of the honey bee populations need further research.     

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.     

Microsporidia infecting Apis mellifera: coexistence or competition. Is Nosema ceranae replacing Nosema apis?

Nosema ceranae has been suggested to be replacing Nosema apis in some populations of Apis mellifera honeybees. However, this replacement from one to the other is not supported when studying the distribution and prevalence of both microsporidia in professional apiaries in Spanish territories (transverse study), their seasonal pattern in experimental hives with co-infection or their prevalence at individual level (either in worker bees or drones). Nevertheless, N.ceranae has shown to present a higher prevalence at all the studied levels that could indicate any advantage for its development over N.apis or that it is more adapted to Spanish conditions. Also, both microsporidia show a different pattern of preference for its development according to the prevalence in the different Spanish bioclimatic belts studied. Finally, the fact that all analyses were carried out using an Internal PCR Control (IPC) newly developed guarantees the confidence of the data extracted from the PCR analyses. This IPC provides a useful tool for laboratory detection of honeybee pathogens.     

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.     

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.     

The effect of induced queen replacement on Nosema spp. infection in honey bee (Apis mellifera iberiensis) colonies

Microsporidiosis of adult honeybees caused by Nosema apis and Nosema ceranae is a common worldwide disease with negative impacts on colony strength and productivity. Few options are available to control the disease at present. The role of the queen in bee population renewal and the replacement of bee losses due to Nosema infection is vital to maintain colony homeostasis. Younger queens have a greater egg laying potential and they produce a greater proportion of uninfected newly eclosed bees to compensate for adult bee losses; hence, a field study was performed to determine the effect of induced queen replacement on Nosema infection in honey bee colonies, focusing on colony strength and honey production. In addition, the impact of long-term Nosema infection of a colony on the ovaries and ventriculus of the queen was evaluated. Queen replacement resulted in a remarkable decrease in the rates of Nosema infection, comparable with that induced by fumagillin treatment. However, detrimental effects on the overall colony state were observed due to the combined effects of stressors such as the queenless condition, lack of brood and high infection rates. The ovaries and ventriculi of queens in infected colonies revealed no signs of Nosema infection and there were no lesions in ovarioles or epithelial ventricular cells.     

A cell culture model for Nosema ceranae and Nosema apis allows new insights into the life cycle of these important honey bee-pathogenic microsporidia

The population of managed honey bees has been dramatically declining in the recent past in many regions of the world. Consensus now seems to be that pathogens and parasites (e.g. the ectoparasitic mite Varroa destructor, the microsporidium Nosema ceranae and viruses) play a major role in this demise. However, little is known about host-pathogen interactions for bee pathogens and attempts to develop novel strategies to combat bee diseases have been hampered by this gap in our knowledge. One reason for this dire situation is the complete lack of cell cultures for the propagation and study of bee pathogens. Here we present a cell culture model for two honey bee-pathogenic microsporidian species, Nosema apis and N. ceranae. Our cell culture system is based on a lepidopteran cell line, which proved to be susceptible to infection by both N. ceranae and N. apis and enabled us to illustrate the entire life cycle of these microsporidia. We observed hitherto undescribed spindle-shaped meronts and confirmed our findings in infected bees. Our cell culture model provides a previously unavailable means to explore the nature of interactions between the honey bee and its pathogen complex at a mechanistic level and will allow the development of novel treatment strategies.     

A new threat to honey bees, the parasitic phorid fly Apocephalus borealis

Honey bee colonies are subject to numerous pathogens and parasites. Interaction among multiple pathogens and parasites is the proposed cause for Colony Collapse Disorder (CCD), a syndrome characterized by worker bees abandoning their hive. Here we provide the first documentation that the phorid fly Apocephalus borealis, previously known to parasitize bumble bees, also infects and eventually kills honey bees and may pose an emerging threat to North American apiculture. Parasitized honey bees show hive abandonment behavior, leaving their hives at night and dying shortly thereafter. On average, seven days later up to 13 phorid larvae emerge from each dead bee and pupate away from the bee. Using DNA barcoding, we confirmed that phorids that emerged from honey bees and bumble bees were the same species. Microarray analyses of honey bees from infected hives revealed that these bees are often infected with deformed wing virus and Nosema ceranae. Larvae and adult phorids also tested positive for these pathogens, implicating the fly as a potential vector or reservoir of these honey bee pathogens. Phorid parasitism may affect hive viability since 77% of sites sampled in the San Francisco Bay Area were infected by the fly and microarray analyses detected phorids in commercial hives in South Dakota and California's Central Valley. Understanding details of phorid infection may shed light on similar hive abandonment behaviors seen in CCD.     

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.     

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.     

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.     

Comparison of within hive sampling and seasonal activity of Nosema ceranae in honey bee colonies

Nosema ceranae is a microsporidian parasite of the European honey bee, Apis mellifera, that is found worldwide and in multiple Apis spp.; however, little is known about the effects of N. ceranae on A. mellifera. Previous studies using spore counts suggest that there is no longer a seasonal cycle for N. ceranae and that it is found year round with little variation in infection intensity among months. Our goal was to determine whether infection levels differ in bees collected from different areas of the hive and if there may be seasonal differences in N. ceranae infections. A multiplex species-specific real-time PCR assay was used for the detection and quantification of N. ceranae. Colonies were sampled monthly from September 2009-2010 by collecting workers from honey supers, the fringe of the brood nest, and the brood nest. We found that all bees sampled were infected with N. ceranae and that there was no significant difference in infection levels among the different groups of bees sampled (P=0.74). However, significant differences in colony infection levels were found at different times of the year (P<0.01) with the highest levels in April-June and lower levels in the fall and winter. While our study was only performed for one year, it sheds light on the fact that there may be a seasonality to N. ceranae infections. Being able to predict future N. ceranae infections can be used to better advise beekeepers on N. ceranae management.     

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.     

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.     

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.     

Cophylogeny of Nosema (Microsporidia: Nosematidae) and bees (Hymenoptera: Apidae) suggests both cospeciation and a host-switch

Some microsporidian parasites belonging to the genus Nosema infect bees. Previous phylogenies of these parasites have produced alternative, conflicting relationships. We analyzed separately, and in combination, large and small subunit ribosomal DNA sequences of Nosema species infecting bees under neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian frameworks. We observed a sister relationship between Nosema ceranae and Nosema bombi, with Nosema apis as a basal member to this group. When compared to their respective hosts (Apis cerana, Bombus spp., and A. mellifera), 2 plausible evolutionary scenarios emerged. The first hypothesis involves a common ancestor of N. bombi host-switching from a historical Bombus lineage to A. cerana. The second suggests an ancestral N. ceranae host-switching to a species of Bombus. The reported events offer insight into the evolutionary history of these organisms and may explain host specificity and virulence of Nosema in these economically important insects.     

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.     

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.     

An experiment on comb orientation by honey bees (Hymenoptera: Apidae) in traditional hives

The orientation of combs in traditional beehives is extremely important for obtaining a marketable honey product. However, the factors that could determine comb orientation in traditional hives and the possibilities of inducing honey bees, Apis mellifera (L.), to construct more desirable combs have not been investigated. The goal of this experiment was to determine whether guide marks in traditional hives can induce bees to build combs of a desired orientation. Thirty-two traditional hives of uniform dimensions were used in the experiment. In 24 hives, ridges were formed on the inner surfaces of the hives with fermented mud to obtain different orientations, circular, horizontal, and spiral, with eight replicates of each treatment. In the remaining eight control hives, the inner surface was left smooth. Thirty-two well-established honey bee colonies from other traditional hives were transferred to the prepared hives. The colonies were randomly assigned to the four treatment groups. The manner of comb construction in the donor and experimental hives was recorded. The results showed that 22 (91.66%) of the 24 colonies in the treated groups built combs along the ridges provided, whereas only 2 (8.33%) did not. Comb orientation was strongly associated with the type of guide marks provided. Moreover, of the 18 colonies that randomly fell to patterns different from those of their previous nests, 17 (94.4%) followed the guide marks provided, irrespective of the comb orientation type in their previous nest. Thus, comb orientation appears to be governed by the inner surface pattern of the nest cavity. The results suggest that even in fixed-comb hives, honey bees can be guided to build combs with orientations suitable to honey harvesting, without affecting the colonies.