Asymmetrical coexistence of Nosema ceranae and Nosema apis in honey bees

Globalization has provided opportunities for parasites/pathogens to cross geographic boundaries and expand to new hosts. Recent studies showed that Nosema ceranae, originally considered a microsporidian parasite of Eastern honey bees, Apis cerana, is a disease agent of nosemosis in European honey bees, Apis mellifera, along with the resident species, Nosema apis. Further studies indicated that disease caused by N. ceranae in European honey bees is far more prevalent than that caused by N. apis. In order to gain more insight into the epidemiology of Nosema parasitism in honey bees, we conducted studies to investigate infection of Nosema in its original host, Eastern honey bees, using conventional PCR and duplex real time quantitative PCR methods. Our results showed that A. cerana was infected not only with N. ceranae as previously reported [Fries, I., Feng, F., Silva, A.D., Slemenda, S.B., Pieniazek, N.J., 1996. Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). Eur. J. Protistol. 32, 356-365], but also with N. apis. Both microsporidia produced single and mixed infections. Overall and at each location alone, the prevalence of N. ceranae was higher than that of N. apis. In all cases of mixed infections, the number of N. ceranae gene copies (corresponding to the parasite load) significantly out numbered those of N. apis. Phylogenetic analysis based on a variable region of small subunit ribosomal RNA (SSUrRNA) showed four distinct clades of N. apis and five clades of N. ceranae and that geographical distance does not appear to influence the genetic diversity of Nosema populations. The results from this study demonstrated that duplex real-time qPCR assay developed in this study is a valuable tool for quantitative measurement of Nosema and can be used to monitor the progression of microsprodian infections of honey bees in a timely and cost efficient manner.     

Does fumagillin control the recently detected invasive parasite Nosema ceranae in western honey bees (Apis mellifera)?

Western honey bee (Apis mellifera) colonies in Nova Scotia, Canada were sampled in spring and late summer 2007 to evaluate efficacy of fumagillin dicyclohexylammonium (hereafter, fumagillin) against Nosema ceranae. Colonies treated with fumagillin in September 2006 (n = 94) had significantly lower Nosema intensity in spring 2007 than did colonies that received no treatment (n = 51), but by late summer 2007 no difference existed between groups. Molecular sequencing of 15 infected colonies identified N. ceranae in 93.3% of cases, suggesting that fumagillin is successful at temporarily reducing this recent invasive parasite in western honey bees.     

Infra-Population and -Community Dynamics of the Parasites Nosema apis and Nosema ceranae,and Consequences for Honey Bee (Apis mellifera) Hosts

Nosema spp. fungal gut parasites are among myriad possible explanations for contemporary increased mortality of western honey bees (Apis mellifera, hereafter honey bee) in many regions of the world. Invasive Nosema ceranae is particularly worrisome because some evidence suggests it has greater virulence than its congener N. apis. N. ceranae appears to have recently switched hosts from Asian honey bees (Apis cerana) and now has a nearly global distribution in honey bees, apparently displacing N. apis. We examined parasite reproduction and effects of N. apis, N. ceranae, and mixed Nosema infections on honey bee hosts in laboratory experiments. Both infection intensity and honey bee mortality were significantly greater for N. ceranae than for N. apis or mixed infections; mixed infection resulted in mortality similar to N. apis parasitism and reduced spore intensity, possibly due to inter-specific competition. This is the first long-term laboratory study to demonstrate lethal consequences of N. apis and N. ceranae and mixed Nosema parasitism in honey bees, and suggests that differences in reproduction and intra-host competition may explain apparent heterogeneous exclusion of the historic parasite by the invasive species.     

Nosema ceranae, a new parasite in Thai honeybees

Adult workers of Apis cerana, Apis florea and Apis mellifera from colonies heavily infected with Nosema ceranae were selected for molecular analyses of the parasite. PCR-specific 16S rRNA primers were designed, cloned, sequenced and compared to GenBank entries. The sequenced products corresponded to N. ceranae. We then infected A. cerana with N. ceranae spores isolated from A. florea workers. Newly emerged bees from healthy colonies were fed 10,000, 20,000 and 40,000 spores/bee. There were significant dosage dependent differences in bee infection and survival rates. The ratio of infected cells to non-infected cells increased at 6, 10 and 14 d post infection. In addition, hypopharyngeal glands of bees from the control group had significantly higher protein concentrations than infected groups. Bees infected with 40,000 spores/bee had the lowest protein concentrations. Thus, N. ceranae isolated from A. florea is capable of infecting another bee species, impairing hypopharyngeal gland protein production and reducing bee survival in A. cerana.     

Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia

Nosema ceranae is a recently described pathogen of Apis mellifera and Apis cerana. Relatively little is known about the distribution or prevalence of N. ceranae in the United States. To determine the prevalence and potential impact of this new pathogen on honey bee colonies in Virginia, over 300 hives were sampled across the state. The samples were analyzed microscopically for Nosema spores and for the presence of the pathogen using real-time PCR. Our studies indicate that N. ceranae is the dominant species in Virginia with an estimated 69.3% of hives infected. Nosema apis infections were only observed at very low levels (2.7%), and occurred only as co-infections with N. ceranae. Traditional diagnoses based on spore counts alone do not provide an accurate indication of colony infections. We found that 51.1% of colonies that did not have spores present in the sample were infected with N. ceranae when analyzed by real-time PCR. In hives that tested positive for N. ceranae, average C_T values were used to diagnose a hive as having a low, moderate, or a heavy infection intensity. Most infected colonies had low-level infections (73%), but 11% of colonies had high levels of infection and 16% had moderate level infections. The prevalence and mean levels of infection were similar in different regions of the state.     

Effects at Nearctic north-temperate latitudes of indoor versus outdoor overwintering on the microsporidium Nosema ceranae and western honey bees (Apis mellifera)

In northern temperate climates, western honey bee (Apis mellifera) colonies can be wintered outdoors exposed to ambient conditions, or indoors in a controlled setting. Because very little is known about how this affects the recently-detected microsporidium Nosema ceranae, we investigated effects of indoor versus outdoor overwintering on spring N. ceranae intensity (spores per bee), and on winter and spring colony mortality. For colonies medicated with Fumagilin-B® to control N. ceranae, overwintering treatment did not affect N. ceranae intensity, despite outdoor-wintered colonies having significantly greater mortality. These findings suggest that N. ceranae may not always pose the most significant threat to western honey bees, and that indoor-wintering may ensure that a greater number of colonies are available for honey production and pollination services during the summer.     

Ecdysteroid titers in pupae of highly social bees relate to distinct modes of caste development

Modifications in endocrine programs are common mechanisms that generate alternative phenotypes. In order to understand how such changes may have evolved, we analyzed the pupal ecdysteroid titers in two closely related, highly social bees: the honey bee, Apis mellifera, and a stingless bee, Melipona quadrifasciata. In both species, the ecdysteroid titers in queens reached their peak levels earlier than in workers. Titer levels at peak maxima did not differ for the honey bee castes, but in Melipona they were twofold higher in queens than in workers. During the second half of pupal development, when the ecdysteroid titers decrease and the cuticle progressively melanizes, the titer in honey bee queens remained higher than in workers, while the reverse situation was observed in Melipona. Application of the juvenile hormone analog Pyriproxyfen^® to spinning-stage larvae of Melipona induced queen development. Endocrinologically this was manifest in a queen-like profile of the pupal ecdysteroid titer. Comparing these data with previous results on preimaginal hormone titers in another stingless bee, we conclude that the timing and height of the pupal ecdysteroid peak may depend on the nature of the specific stimuli that initially trigger diverging queen/worker development. In contrast, the interspecific differences in the late pupal ecdysteroid titer profiles mainly seem to be related to caste-specific programs in tissue differentiation, including cuticle pigmentation.     

Infections of Nosema ceranae in four different honeybee species

The microsporidium Nosema ceranae is detected in honeybees in Thailand for the first time. This endoparasite has recently been reported to infect most Apis mellifera honeybee colonies in Europe, the US, and parts of Asia, and is suspected to have displaced the endemic endoparasite species, Nosema apis, from the western A. mellifera. We collected and identified species of microsporidia from the European honeybee (A. mellifera), the cavity nesting Asian honeybee (Apis cerana), the dwarf Asian honeybee (Apis florea) and the giant Asian honeybee (Apis dorsata) from colonies in Northern Thailand. We used multiplex PCR technique with two pairs of primers to differentiate N. ceranae from N. apis. From 80 A. mellifera samples, 62 (77.5%) were positively identified for the presence of the N. ceranae. Amongst 46 feral colonies of Asian honeybees (A. cerana, A. florea and A. dorsata) examined for Nosema infections, only N. ceranae could be detected. No N. apis was found in our samples. N. ceranae is found to be the only microsporidium infesting honeybees in Thailand. Moreover, we found the frequencies of N. ceranae infection in native bees to be less than that of A. mellifera.     

Nosema ceranae has been present in Brazil for more than three decades infecting Africanized honey bees

Until the mid-1990s, the only microsporidium known to infect bees of the genus Apis was Nosema apis. A second species, Nosema ceranae, was first identified in 1996 from Asian honey bees; it is postulated that this parasite was transmitted from the Asian honey bee, Apis cerana, to the European honey bee, Apis mellifera. Currently, N. ceranae is found on all continents and has often been associated with honey bee colony collapse and other reports of high bee losses. Samples of Africanized drones collected in 1979, preserved in alcohol, were analyzed by light microscopy to count spores and were subjected to DNA extraction, after which duplex PCR was conducted. All molecular analyses (triplicate) indicated that the drones were infected with both N. ceranae and N. apis. PCR products were sequenced and matched to sequences reported in the GenBank (Acc. Nos. JQ639316.1 and JQ639301.1). The venation pattern of the wings of these males was compared to those of the current population living in the same area and with the pattern of drones collected in 1968 from Ribeirão Preto, SP, Brazil, from a location close to where African swarms first escaped in 1956. The morphometric results indicated that the population collected in 1979 was significantly different from the current living population, confirming its antiquity. Considering that the use of molecular tools for identifying Nosema species is relatively recent, it is possible that previous reports of infections (which used only light microscopy, without ultrastructural analysis) wrongly identified N. ceranae as N. apis. Although we can conclude that N. ceranae has been affecting Africanized honeybees in Brazil for at least 34 years, the impact of this pathogen remains unclear.     

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.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

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.     

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.     

Sex-Specific Differences in Pathogen Susceptibility in Honey Bees (Apis mellifera)

Sex-related differences in susceptibility to pathogens are a common phenomenon in animals. In the eusocial Hymenoptera the two female castes, workers and queens, are diploid and males are haploid. The haploid susceptibility hypothesis predicts that haploid males are more susceptible to pathogen infections compared to females. Here we test this hypothesis using adult male (drone) and female (worker) honey bees (Apis mellifera), inoculated with the gut endoparasite Nosema ceranae and/or black queen cell virus (BQCV). These pathogens were chosen due to previously reported synergistic interactions between Nosema apis and BQCV. Our data do not support synergistic interactions between N. ceranae and BQCV and also suggest that BQCV has limited effect on both drone and worker health, regardless of the infection level. However, the data clearly show that, despite lower levels of N. ceranae spores in drones than in workers, Nosema-infected drones had both a higher mortality and a lower body mass than non-infected drones, across all treatment groups, while the mortality and body mass of worker bees were largely unaffected by N. ceranae infection, suggesting that drones are more susceptible to this pathogen than workers. In conclusion, the data reveal considerable sex-specific differences in pathogen susceptibility in honey bees and highlight the importance of ultimate measures for determining susceptibility, such as mortality and body quality, rather than mere infection levels.     

Patriline differences in emergency queen rearing in the honey bee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

Summary In the polyandrous honey bee, Apis mellifera, workers can potentially increase their inclusive fitness by rearing full-sister queens. If the mother queen dies suddenly, workers feed a few larvae in worker cells with royal jelly and rear them into queens (emergency queen rearing). Using DNA microsatellite markers we determined the patriline of emergency queens reared in two colonies headed by naturally-mated queens before being made queenless. We found that some patrilines were reared more than others in one colony, but not in the other. These differences between colonies suggest that selective rearing is not always present and this might explain the mixed results of previous nepotism studies in the honey bee.Received 10 February 2003; revised 7 March 2003; accepted 17 March 2003.     

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.     

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

Nosema ceranae is an intracellular microsporidian parasite of the Asian honey bee Apis cerana and the European honey bee Apis mellifera. Until relatively recently, A. mellifera honey bees were naïve to N. ceranae infection. Symptoms of nosemosis, or Nosema disease, in the infected hosts include immunosuppression, damage to gut epithelium, nutrient and energetic stress, precocious foraging and reduced longevity of infected bees. Links remain unclear between immunosuppression, the symptoms of nutrient and energetic stress, and precocious foraging behavior of hosts. To clarify physiological connections, we inoculated newly emerged A. mellifera adult workers with N. ceranae spores, and over 21?days post inoculation (21?days?pi), gauged infection intensity and quantified expression of genes representing two innate immune pathways, Toll and Imd. Additionally, we measured each host’s whole-body protein, lipids, carbohydrates and quantified respirometric and activity levels. Results show sustained suppression of genes of both humorally regulated immune response pathways after 6?days?pi. At 7?days?pi, elevated protein levels of infected bees may reflect synthesis of antimicrobial peptides from an initial immune response, but the lack of protein gain compared with uninfected bees at 14?days?pi may represent low de novo protein synthesis. Carbohydrate data do not indicate that hosts experience severe metabolic stress related to this nutrient. At 14?days?pi infected honey bees show high respirometric and activity levels, and corresponding lipid loss, suggesting lipids may be used as fuel for increased metabolic demands resulting from infection. Accelerated lipid loss during nurse honey bee behavioral development can have cascading effects on downstream physiology that may lead to precocious foraging, which is a major factor driving colony collapse.     

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.     

Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand

Nosema ceranae was found to infect four different host species including the European honeybee (A. mellifera) and the Asian honeybees (Apis florea, A. cerana and Apis dorsata) collected from apiaries and forests in Northern Thailand. Significant sequence variation in the polar tube protein (PTP1) gene of N. ceranae was observed with N. ceranae isolates from A. mellifera and A. cerana, they clustered into the same phylogenetic lineage. N. ceranae isolates from A. dorsata and A. florea were grouped into two other distinct clades. This study provides the first elucidation of a genetic relationship among N. ceranae strains isolated from different host species and demonstrates that the N. ceranae PTP gene was shown to be a suitable and reliable marker in revealing genetic relationships within species.