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.     

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.     

Calcofluor White M2R与Sytox Green双重染色法鉴别蜜蜂微孢子虫

东方蜜蜂微孢子虫(Nosema ceranae)是一种广泛寄生于东方蜜蜂Apis cerana,西方蜜蜂Apis mellifera和熊蜂Bombus Latreille上的寄生虫,对蜜蜂和熊蜂的危害较大,进而影响养蜂业的发展。本实验采用荧光染色试剂Calcofluor White M2R与核酸染料Sytox Green双重染法来鉴别蜜蜂或熊蜂体内的N.ceranae及孢子的存活状态。结果得出,在荧光显微镜下可见死孢子被染上黄绿色荧光,活的呈现蓝白色荧光,而寄主细胞、细菌、病毒等不被染色。这是一种快速有效鉴别N.ceranae及其死活的方法,从而判定蜜蜂或熊蜂体内的微孢子虫在是否具有侵染活性,对微孢子虫的研究及药物防治具有重要作用。     

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.     

Further evidence of an oriental origin for Nosema ceranae (Microsporidia: Nosematidae)

Although Nosema ceranae was first isolated from the Asian honeybee (Apis cerana) in Asia and then subsequently recognized as a widespread gut parasite of the Western honeybee (Apis mellifera), its origins and primary host are yet to be accurately established. In this study we examined the possibility of an Asian origin for the parasite by looking for evidence of its ongoing spread out of Asia. To do this, we surveyed for the presence of N. ceranae in A. cerana and A. mellifera on isolated islands of the Solomon Islands (Pacific region), most of which were inhabited with A. mellifera that had been introduced from Australia and New Zealand at a time when N. ceranae was not present in either country, but on which some had also recently become inhabited with invasive A. cerana that originated from Asia with no prior history of contact with A. mellifera infected with N. ceranae. We also sought to verify previous findings that N. ceranae was widespread in Asian honeybees by surveying for its presence in isolated populations of the Asian honeybees, A. cerana, A. koschevnikovi, A. nigrocincta and A. florea. We obtained evidence that A. cerana introduced N. ceranae to A. mellifera in the Solomon Islands and also confirmed the widespread occurrence of the parasite in Asian honeybees, even reporting it for the first time in A. koschevnikovi from Borneo. Our findings provide further support for the hypothesis that N. ceranae has only recently emerged from Asia to become a parasite of A. mellifera.     

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.     

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,...     

Prevalence of Nosema species infections in Apis cerana japonica and Apis mellifera honeybees in the Tohoku region of Japan

We investigated here, the prevalence of Nosema microsporidia infections in the honeybees, Apis cerana japonica and Apis mellifera, in the Tohoku region of Japan. We detected Nosema ceranae DNA in 14 (2.8%) of 509 A. cerana japonica and in 34 (21.9%) of 155 A. mellifera honeybees from Aomori, Iwate, Akita, Yamagata, and Fukushima prefectures. Nosema apis DNA was undetectable in A. cerana japonica and A. mellifera. The unidentifiable Nosema species that genetically differed from N. apis, N. ceranae, and N. neumanni in terms of small subunit (SSU) rDNA, large subunit rDNA, and internal transcribed spacer sequences was identified in 105 (20.6%) of 509 A. cerana japonica and in 1 (0.6%) of 155 A. mellifera honeybees, and from Iwate prefecture. A phylogenetic tree based on SSU rDNA sequences showed that the Nosema sp. belonged to the same clade as N. thomsoni detected in moth and solitary bees in North America and N. pieriae found in cabbage butterfly in Turkey, which have not hitherto been detected in honeybees. The morphological characteristics of the spores should be analyzed to enable species identification of the Nosema sp.     

蜜蜂微孢子虫在中国的自然种系构成初探

蜜蜂微孢子虫(Nosema apis及Nosema ceranae)是微孢子虫的典型代表之一,由它寄生蜜蜂所产生的疾病,称为蜜蜂微孢子虫病。通过目前国际上普遍使用的克隆16srRNA基因(16srDNA)的片段并测序的方法来进行蜜蜂微孢子虫在我国主要养蜂地区的分布情况。结果表明,在我国主要养蜂地区,造成大量西方蜜蜂患蜜蜂微孢子虫病的是东方蜜蜂微孢子虫Nosema ceranae,至今为止未发现过去我们广泛认定的西方蜜蜂微孢子虫Nosema apis。     

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.     

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.     

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.     

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.     

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.     

中华蜜蜂mrjp1 cDNA的克隆及其序列分析

构建中华蜜蜂(Apis cerana cerana)8日龄工蜂头部cDNA文库,利用中蜂基因组的mrjp3部分基因片段作为杂交探针,采用DIG标记筛选cDNA文库,获得mrjps阳性克隆120个;对阳性克隆进行PCR扩增和测序,通过NCBI的BLAST序列比对,获得12个与印度蜂(Apis cerana india)、西方蜜蜂(Apis mellifera L.)mrjp1基因同源的中蜂mtjp1 cDNA片段,并进一步对中华蜜蜂mrjp1的cDNA全序列进行测定和分析。序列比对分析表明,东方蜜蜂(Apis cerana)与西方蜜蜂mrjp1的cDNA序列相似性为93．78％,中华蜜蜂与印度蜂的相似性高达99．36％,这一结果从分子水平证实中华蜜蜂与印度蜂有较近的共同祖先,而东方蜜蜂与西方蜜蜂的亲缘关系较远。     

Polymorphism and recombination for rDNA in the putatively asexual microsporidian Nosema ceranae, a pathogen of honeybees

Nosema ceranae is currently one of the major pathogens of honeybees, related to the worldwide colony losses phenomenon. The genotyping of strains based on ribosomal DNA (rDNA) can be misleading if the repeated units are not identical. The analysis of cloned rDNA fragments containing the intergenic spacer (IGS) and part of the rDNA small-subunit (SSU) gene, from N. ceranae isolates from different European and Central Asia populations, revealed a high diversity of sequences. The variability involved single-nucleotide polymorphisms and insertion/deletions, resulting in 79 different haplotypes. Two sequences from the same isolate could be as different as any pair of sequences from different samples; in contrast, identical haplotypes were also found in very different geographical origins. Consequently, haplotypes cannot be organized in a consistent phylogenetic tree, clearly indicating that rDNA is not a reliable marker for the differentiation of N. ceranae strains. The results indicate that recombination between different sequences may produce new variants, which is quite surprising in microsporidia, usually considered to have an asexual mode of reproduction. The diversity of sequences and their geographical distribution indicate that haplotypes of different lineages may occasionally be present in a same cell and undergo homologue recombination, therefore suggesting a sexual haplo-diploid cycle.     

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.