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Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae-infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.

This study examined the control of nosemosis caused by Nosema ceranae, one of the hard-to-control diseases of honey bees, using RNA interference (RNAi) technology. Double-stranded RNA (dsRNA) for RNAi application targeted the mitosome-related genes of N. ceranae. Among the various mitosome-related genes, NCER_100882, NCER_101456, NCER_100157, and NCER_100686 exhibited relatively low homologies with the orthologs of Apis mellifera. Four gene-specific dsRNAs were prepared against the target genes and applied to the infected A. mellifera to analyze Nosema proliferation and honey bee survival. Two dsRNAs specifics to NCER_101456 and NCER_100157 showed high inhibitory effects on spore production by exhibiting only 62% and 67%, respectively, compared with the control. In addition, these dsRNA treatments significantly rescued the honey bees from the fatal nosemosis. It was confirmed that the inhibition of Nosema spore proliferation and the increase in the survival rate of honey bees were resulted from a decrease in the expression level of each target gene by dsRNA treatment. However, dsRNA mixture treatment was no more effective than single treatments in the rescue from the nosemosis. It is expected that the four newly identified mitosome-related target genes in this study can be effectively used for nosemosis control using RNAi technology., (© 2020 Wiley Periodicals LLC.)

Background: Gut microbial communities can contribute positively and negatively to host health. So far, eight core bacterial taxonomic clusters have been reported in honey bees. These bacteria are involved in host metabolism and defenses. Nosema ceranae is a gut intracellular parasite of honey bees which destroys epithelial cells and gut tissue integrity. Studies have shown protective impacts of honey bee gut microbiota towards N. ceranae infection. However, the impacts of N. ceranae on the relative abundance of honey bee gut microbiota remains unclear, and has been confounded during prior infection assays which resulted in the co-inoculation of bacteria during Nosema challenges. We used a novel method, the suppression of N. ceranae with specific siRNAs, to measure the impacts of Nosema on the gut microbiome., Results: Suppressing N. ceranae led to significant positive effects on microbial abundance. Nevertheless, 15 bacterial taxa, including three core taxa, were negatively correlated with N. ceranae levels. In particular, one co-regulated group of 7 bacteria was significantly negatively correlated with N. ceranae levels., Conclusions: N. ceranae are negatively correlated with the abundance of 15 identified bacteria. Our results provide insights into interactions between gut microbes and N. ceranae during infection.

The health of honey bees is threatened by multiple factors, including viruses and parasites. We screened 557 honey bee ( Apis mellifera ) colonies from 155 beekeepers distributed all over Belgium to determine the prevalence of seven widespread viruses and two parasites ( Varroa sp. and Nosema sp.). Deformed wing virus B (DWV-B), black queen cell virus (BQCV), and sacbrood virus (SBV) were highly prevalent and detected by real-time RT-PCR in more than 95% of the colonies. Acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and deformed wing virus A (DWV-A) were prevalent to a lower extent (between 18 and 29%). Most viruses were only present at low or moderate viral loads. Nevertheless, about 50% of the colonies harbored at least one virus at high viral load (>10 7 genome copies/bee). Varroa mites and Nosema sp. were found in 81.5% and 59.7% of the honey bee colonies, respectively, and all Nosema were identified as Nosema ceranae by real time PCR. Interestingly, we found a significant correlation between the number of Varroa mites and DWV-B viral load. To determine the combined effect of these and other factors on honey bee health in Belgium, a follow up of colonies over multiple years is necessary.

The reduction of bumblebee populations has been reported in the last decades, and the microsporidian parasite Nosema bombi is considered as one of the factors contributing to such reduction. Although the decline of bee populations affects both wild plants and human food supply, the effects of Nosema spp. infections are not known because it is difficult to obtain infective spores from wild bees due to their low prevalence. Microscopical observation of fecal samples or midgut homogenates and/or PCR are generally used for N. bombi detection. However, the germination rate of microsporidian spore declines if they are kept at 4 °C for a long time or frozen. It is therefore crucial to minimize the diagnosis and isolation time of infective spores from field-collected samples. Therefore, we performed a loop-mediated isothermal amplification (LAMP) assay for the direct detection of N. bombi in bumblebee midgut homogenates. Using this method, we could detect N. bombi from individuals from which it was visible under the microscope and directly from wild individuals.

Nosema ceranae is the most prevalent endoparasite of Apis mellifera iberiensis and it is a major health problem for bees worldwide. The infective capacity of N. ceranae has been demonstrated experimentally in honey bee brood, however no data are available about its prevalence in brood under natural conditions. Thus, brood combs from 10 different hives were analyzed over two consecutive years, taking samples before and after winter. A total of 1433 larvae/pupae were analyzed individually and N. ceranae (3.53%) was the microsporidian most frequently detected, as opposed to Nosema apis (0.42%) which was more frequently detected in conjunction with N. ceranae (0.71%). The active multiplication of both microsporidians was confirmed by the expression (real-time-PCR) of the N. ceranae polar tube protein 3 gene and/or the N. apis RNA polymerase II gene in 24% of the brood samples positive for Nosema spp. Both genes are related to microsporidian multiplication. As such, N. ceranae multiplication was confirmed in 1.06% of the samples, while N. apis multiplication was only observed in co-infections with N. ceranae (0.07%). Brood cells were analyzed for the presence of Nosema spp., as those are the immediate environment where the brood stages develop. The brood samples infected by Nosema spp. were in brood cells in which that microsporidians were not detected, while brood cells positive for N. ceranae hosted brood stages that were not apparently infected, indicating that this is unlikely to be the main pathway of infection. Finally, the colonies with brood infected by N. ceranae showed higher levels (numbers) of infected adult bees, although the differences were not significant before (P = 0.260), during (P = 0.055) or after (P = 0.056) brood sampling. These results show that N. ceranae is a bee parasite ubiquitous to all members of the colony, irrespective of the age of the bee. It is also of veterinary interest and should be considered when studying the epidemiology of the disease., (Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The recent global decline in Western honeybee (Apis mellifera) populations is of great concern for pollination and honey production worldwide. Declining honeybee populations are frequently infected by the microsporidian pathogen Nosema ceranae. This species was originally described in the Asiatic honeybee (Apis cerana), and its identification in global A. mellifera hives could result from a recent host transfer. Recent genome studies have found that global populations of this parasite are polyploid and that humans may have fueled their global expansion. To better understand N. ceranae biology, we investigated its genetic diversity within part of their native range (Thailand) and among different hosts (A. mellifera, A. cerana) using both PCR and genome-based methods. We find that Thai N. ceranae populations share many SNPs with other global populations and appear to be clonal. However, in stark contrast with previous studies, we found that these populations also carry many SNPs not found elsewhere, indicating that these populations have evolved in their current geographic location for some time. Our genome analyses also indicate the potential presence of diploidy within Thai populations of N. ceranae.

Two species of microsporidia, Nosema apis and Nosema ceranae, occur regularly and cause significant losses in apiculture throughout the world. N. ceranae is thought to be an emerging pathogen of the European honey bee which is replacing N. apis. Microscopic analysis of honey bees collected in Tyumen region, South-Western Siberia, suggested presence of two microsporidial pathogens slightly differing in spore size and shape. PCR detection using species-specific primer sets 312APIS and 218MITOC followed by PCR product sequencing confirmed the diagnosis of N. apis and N. ceranae, respectively. Microsporidia were present in private apiaries through 2008-2010, and among 21 colonies from 7 localities, two colonies were infected with both pathogens, while infections with N. apis only were detected in 8, and with N. ceranae only in 13 colonies. These data suggest that N. ceranae is widely spread in South-Western Siberia alongside with N. apis and is able to persist in the regions with average January temperatures below -18°C.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene and is conserved in a wide range of eukaryotic organisms. The RNAi mechanism has provided unique opportunities for combating honey bee diseases caused by various parasites and pathogens. Nosema ceranae is a microsporidian parasite of European honey bees, Apis mellifera , and has been associated with honey bee colony losses in some regions of the world. Here we explored the possibility of silencing the expression of a N. ceranae putative virulence factor encoding polar tube protein 3 ( ptp3 ) which is involved in host cell invasion as a therapeutic strategy for controlling Nosema parasites in honey bees. Our studies showed that the oral ingestion of a dsRNA corresponding to the sequences of N. ceranae ptp3 could effectively suppress the expression of the ptp3 gene in N. ceranae -infected bees and reduce Nosema load. In addition to the knockdown of ptp3 gene expression, ingestion of ptp3 -dsRNA also led to improved innate immunity in bees infected with N. ceranae along with an improvement in physiological performance and lifespan compared with untreated control bees. These results strongly suggest that RNAi-based therapeutics hold real promise for the effective treatment of honey bee diseases in the future, and warrant further investigation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Nosema ceranae is now a widespread honey bee pathogen with high incidence in apiculture. Rapid and reliable detection and quantification methods are a matter of concern for research community, nowadays mainly relying on the use of biomolecular techniques such as PCR, RT-PCR or HRMA. The aim of this technical paper is to provide a new qPCR assay, based on the highly-conserved protein coding gene Hsp70, to detect and quantify the microsporidian Nosema ceranae affecting the western honey bee Apis mellifera. The validation steps to assess efficiency, sensitivity, specificity and robustness of the assay are described also., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Nosema ceranae is a hot topic in honey bee health as reflected by numerous papers published every year. This review presents an update of the knowledge generated in the last 12 years in the field of N. ceranae research, addressing the routes of transmission, population structure and genetic diversity. This includes description of how the infection modifies the honey bee's metabolism, the immune response and other vital functions. The effects on individual honey bees will have a direct impact on the colony by leading to losses in the adult's population. The absence of clear clinical signs could keep the infection unnoticed by the beekeeper for long periods. The influence of the environmental conditions, beekeeping practices, bee genetics and the interaction with pesticides and other pathogens will have a direct influence on the prognosis of the disease. This review is approached from the point of view of the Mediterranean countries where the professional beekeeping has a high representation and where this pathogen is reported as an important threat., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Nosema ceranae causes widespread infection in adult workers of European honey bees, Apis mellifera, and has often been linked to honey bee colony losses worldwide. Previous investigations of honey bee immune response to N. ceranae infection were largely based on laboratory experiment, however, little is known about the immune response of honey bees that are naturally infected by N. ceranae. Here, we compared the infection levels of N. ceranae in three different categories of adult bees (emergent bees, nurses, and foragers) and detected the host immune response to the N. ceranae infection under natural conditions. Our studies showed that the Nosema spore load and infection prevalence varied among the different types of adult workers, and both of them increased as honey bees aged: No infection was detected in emergent bees, nurses had a medium spore load and prevalence, while foragers were with the highest Nosema infection level and prevalence. Quantification of the mRNA levels of antimicrobial peptides (abaecin, apidaecin, defensin-1, defensin-2, and hymenoptaecin) and microbial recognition proteins (PGRP-S1, PGRP-S2, PGRP-S3, PGRP-LC, GNBP1-1, and GNBP1-2) confirmed the involvement of the Toll and/or Imd immune pathways in the host response to N. ceranae infection, and revealed an activation of host immune response by N. ceranae infection under natural conditions. Additionally, the levels of immune response were positively correlated with the Nosema spore loads in the infected bees. The information gained from this study will be relevant to the predictive modeling of honey bee disease dynamics for Nosema disease prevention and management.

Besides the incipient research effort, the role of parasites as drivers of the reduction affecting pollinator populations is mostly unknown. Given the worldwide extension of the beekeeping practice and the diversity of pathogens affecting Apis mellifera populations, honey bee colonies are a certain source of parasite dispersion to other species. Here, we communicate the detection of the microsporidium Nosema ceranae, a relatively new parasite of honey bees, in stingless bees (Meliponini) and the social wasp Polybia scutellaris (Vespidae) samples from Argentina and Brazil by means of duplex PCR. Beyond the geographic location of the nests, N. ceranae was detected in seven from the eight Meliponini species analyzed, while Nosema apis, another common parasite of A. mellifera, was absent in all samples tested. Further research is necessary to determine if the presence of the parasite is also associated with established infection in host tissues. The obtained information enriches the current knowledge about pathologies that can infect or, at least, be vectored by native wild pollinators from South America.

The microsporidium Nosema neumanni n. sp., a new parasite of the honeybee Apis mellifera is described based on its ultra-structural and molecular characteristics. Structures resembling microsporidian spores were found by microscopic examination of honeybees from Uganda. Molecular confirmation failed when PCR primers specific for Nosema apis and Nosema ceranae were used, but was successful with primers covering the whole family of Nosematidae. We performed transmission electron microscopy and found typical microsporidian spores which were smaller (length: 2.36±0.14μm and width: 1.78±0.06μm; n=6) and had fewer polar filament coils (10-12) when compared to those of known species infecting honeybees. The entire 16S SSU rRNA region was amplified, cloned and sequenced and was found to be unique with the highest resemblance (97% identity) to N. apis. The incidence of N. neumanni n. sp. in Ugandan honeybees was found to be much higher than of the two other Nosema species., (Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The Western honey bee ( Apis mellifera ) is widely used as commercial pollinator in worldwide agriculture and, therefore, plays an important role in global food security. Among the parasites and pathogens threatening health and survival of honey bees are two species of microsporidia, Nosema apis and Nosema ceranae. Nosema ceranae is considered an emerging pathogen of the Western honey bee. Reports on the spread of N. ceranae suggested that this presumably highly virulent species is replacing its more benign congener N. apis in the global A. mellifera population. We here present a 12 year longitudinal cohort study on the prevalence of N. apis and N. ceranae in Northeast Germany. Between 2005 and 2016, a cohort of about 230 honey bee colonies originating from 23 apiaries was sampled twice a year (spring and autumn) resulting in a total of 5,600 bee samples which were subjected to microscopic and molecular analysis for determining the presence of infections with N. apis or/and N. ceranae . Throughout the entire study period, both N. apis - and N. ceranae -infections could be diagnosed within the cohort. Logistic regression analysis of the prevalence data demonstrated a significant increase of N. ceranae -infections over the last 12 years, both in autumn (reflecting the development during the summer) and in spring (reflecting the development over winter) samples. Cell culture experiments confirmed that N. ceranae has a higher proliferative potential than N. apis at 27° and 33°C potentially explaining the increase in N. ceranae prevalence during summer. In autumn, characterized by generally low infection prevalence, this increase was accompanied by a significant decrease in N. apis -infection prevalence. In contrast, in spring, the season with a higher prevalence of infection, no significant decrease of N. apis infections despite a significant increase in N. ceranae infections could be observed. Therefore, our data do not support a general advantage of N. ceranae over N. apis and an overall replacement of N. apis by N. ceranae in the studied honey bee population.

In recent years, large-scale colony losses of honey bees (Apis mellifera) have been reported and the infection with the microsporidia Nosema ceranae has been involved. However, the effect of N. ceranae at the colony level and its role in colony losses vary in different geographic areas. This difference may be related to the presence of multiple N. ceranae genetic variants resulting in different biological consequences. In this study, we analyzed the genetic diversity of 75 N. ceranae samples obtained from 13 countries and Hawaii through inter-sequence single repetition (ISSR) and evaluated if two of these genetic variants triggered different immune responses when infecting Apis mellifera iberiensis. The genetic diversity analysis showed that 41% of the samples had the same DNA amplification pattern, including samples from most European countries except Spain, while the remaining samples showed high variability. Infection assays were performed to analyze the infection levels and the immune response of bees infected with N. ceranae from Spain and Uruguay. The infected bees presented similar infection levels, and both isolates downregulated the expression of abaecin, confirming the ability of the microsporidia to depress the immune response. Only N. ceranae from Uruguay downregulated the expression level of imd compared to control bees. On the other hand, both genetic variants triggered different expression levels of lysozyme. As imd and lysozyme play important roles in the response to pathogens, these results could reflect differences in the biological consequences of N. ceranae variants in A. mellifera infection.

Host manipulation is a common strategy by parasites to reduce host defense responses, enhance development, host exploitation, reproduction and, ultimately, transmission success. As these parasitic modifications can reduce host fitness, increased selection pressure may result in reciprocal adaptations of the host. Whereas the majority of studies on host manipulation have explored resistance against parasites (i.e. ability to prevent or limit an infection), data describing tolerance mechanisms (i.e. ability to limit harm of an infection) are scarce. By comparing differential protein abundance, we provide evidence of host-parasite interactions in the midgut proteomes of N. ceranae-infected and uninfected honey bees from both Nosema-tolerant and Nosema-sensitive lineages. We identified 16 proteins out of 661 protein spots that were differentially abundant between experimental groups. In general, infections of Nosema resulted in an up-regulation of the bee's energy metabolism. Additionally, we identified 8 proteins that were differentially abundant between tolerant and sensitive honey bees regardless of the Nosema infection. Those proteins were linked to metabolism, response to oxidative stress and apoptosis. In addition to bee proteins, we also identified 3 Nosema ceranae proteins. Interestingly, abundance of two of these Nosema proteins were significantly higher in infected Nosema-sensitive honeybees relative to the infected Nosema-tolerant lineage. This may provide a novel candidate for studying the molecular interplay between N. ceranae and its honey bee host in more detail., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A total of 361 colonies from 59 apiaries located in two temperate and three subtropical eco-regions were examined during the post-harvest period to determine distribution and prevalence of Nosema spp. Apiaries from subtropical eco-regions showed a lower spore count than those from temperate eco-regions. Pure N. ceranae and co-infection were detected in apiaries from all regions. In contrast, pure N. apis infection was exclusively observed in the subtropical study region. The predominant detection of N. apis in a subtropical region joining a southern temperate region where mainly co-infected apiaries were identified is in contrast to previous reports., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: There is great controversy as to whether Microsporidia undergo a sexual cycle. In the paradigmatic case of Nosema ceranae, although there is no morphological evidence of sex, some meiosis-specific genes are present in its reduced genome and there is also high intraspecific variability, with incongruent phylogenies having been systematically obtained. The possibility of sexual recombination is important from an epidemiological standpoint, particularly as N. ceranae is considered to be a major factor in the current disquieting epidemic of widespread bee colony losses. This parasite apparently originated in oriental honey bees, spreading out of Asia and Australia to infect honey bees worldwide. This study had three main objectives: i) to obtain genetic markers that are not part of known multi-copy arrays for strain determination; ii) to shed light on the intraspecific variability and recombination of N. ceranae; and iii) to assess the variability in N. ceranae populations. The answers to these questions are critical to understand the capacity of adaptation of microsporidia., Results: Biallelic polymorphisms were detected at a number of specific points in the five coding loci analyzed from European and Australian isolates of N. ceranae. Heterozygous genotypes were abundant and cloning experiments demonstrate that they reflect the existence of multiple alternative sequences in each isolate. The comparisons of different clones and genotypes clearly indicate that new haplotypes are generated by homologous recombination., Conclusions: The N. ceranae isolates from honey bees correspond to genotypically distinct populations, revealing that individual honey bees may not be infected by a particular clone but rather, a pool of different strains. Homologous recombination implies the existence of a cryptic sex cycle yet to be described in N. ceranae. There are no diagnostic alleles associated with Australian or European origins, nor are there differences between the two hosts, A. cerana and A. mellifera, supporting the absence of biological barriers for N. ceranae transmission. Diversity is high among microsporidia of both these origins, and the maintenance of a high heterozygosis in the recently invaded European populations, could hypothetically underlie the stronger virulence of N. ceranae observed in A. mellifera.

Background: Nosema ceranae infection not only damages honey bee (Apis melifera) intestines, but we believe it may also affect intestinal yeast development and its seasonal pattern. In order to check our hypothesis, infection intensity versus intestinal yeast colony forming units (CFU) both in field and cage experiments were studied., Methods/findings: Field tests were carried out from March to October in 2014 and 2015. N. ceranae infection intensity decreased more than 100 times from 7.6 x 108 in March to 5.8 x 106 in October 2014. A similar tendency was observed in 2015. Therefore, in the European eastern limit of its range, N. ceranae infection intensity showed seasonality (spring peak and subsequent decline in the summer and fall), however, with an additional mid-summer peak that had not been recorded in other studies. Due to seasonal changes in the N. ceranae infection intensity observed in honey bee colonies, we recommend performing studies on new therapeutics during two consecutive years, including colony overwintering. A natural decrease in N. ceranae spore numbers observed from March to October might be misinterpreted as an effect of Nosema spp. treatment with new compounds. A similar seasonal pattern was observed for intestinal yeast population size in field experiments. Furthermore, cage experiments confirmed the size of intestinal yeast population to increase markedly together with the increase in the N. ceranae infection intensity. Yeast CFUs amounted to respectively 2,025 (CV = 13.04) and 11,150 (CV = 14.06) in uninfected and N. ceranae-infected workers at the end of cage experiments. Therefore, honey bee infection with N. ceranae supported additional opportunistic yeast infections, which may have resulted in faster colony depopulations., Competing Interests: The authors have declared that no competing interests exist.

Infestation of bee colonies and apiaries by representatives of the genus Nosema, microsporidian protozoans of European honeybees (Apis mellifera L.), causing nosemosis, in Tomsk Province was investigated. In 2012—2015, nosemosis was detected in 32 out of 124 honeybee colonies (31.3 %) and in 20 out of 64 studied apiaries (25.8 %). The maximal infestation rate of bee colonies and apiaries constituted more than 40 % in 2014—2015. N. apis pathogen was registered in 84.4 % of infected bee colonies (16 apiaries); N. ceranae was identified in 9.4 % of infected bee colonies (2 apiaries); and co-infection (N. apis and N. ceranae) was detected in 6.3 % of infected bee colonies (2 apiaries). The reasons of the spreading of the nosemosis, such as climatic conditions, control of imported bee colonies on the presence of Nosema infection, and some others are discussed.

We previously found transcripts encoding Dicer and Argonaute which are involved in the production of microRNAs, in the honey bee parasite Nosema ceranae. In order to identify microRNAs in N. ceranae, we sequenced small RNAs from midgut tissues of infected honey bees at 24 h intervals for 6 days post infection, covering the complete reproduction cycle for this intracellular parasite. We predicted six microRNA-like small RNAs, all of which were confirmed via RT-qPCR assays. This is the first evidence for microRNA-like small RNAs generated by a microsporidian species, providing new insights into host-parasite interactions involving this widespread taxonomic group., (Published by Elsevier Inc.)

The study was conducted to investigate the effect of Lactobacillus rhamnosus (a commercial probiotic) and inulin (a prebiotic) on the survival rates of honeybees infected and uninfected with Nosema ceranae, the level of phenoloxidase (PO) activity, the course of nosemosis, and the effect on the prevention of nosemosis development in bees. The cells of L. rhamnosus exhibited a high rate of survival in 56.56 % sugar syrup, which was used to feed the honeybees. Surprisingly, honeybees fed with sugar syrup supplemented with a commercial probiotic and a probiotic + prebiotic were more susceptible to N. ceranae infection, and their lifespan was much shorter. The number of microsporidian spores in the honeybees fed for 9 days prior to N. ceranae infection with a sugar syrup supplemented with a commercial probiotic was 25 times higher (970 million spores per one honeybee) than in a control group fed with pure sucrose syrup (38 million spores per one honeybee). PO activity reached its highest level in the hemolymph of this honeybee control group uninfected with N. ceranae. The addition of probiotics or both probiotics and prebiotics to the food of uninfected bees led to the ~2-fold decrease in the PO activity. The infection of honeybees with N. ceranae accompanied an almost 20-fold decrease in the PO level. The inulin supplemented solely at a concentration of 2 μg/mL was the only administrated factor which did not significantly affect honeybees' survival, the PO activity, or the nosemosis infection level. In conclusion, the supplementation of honeybees' diet with improperly selected probiotics or both probiotics and prebiotics does not prevent nosemosis development, can de-regulate insect immune systems, and may significantly increase bee mortality.

This study was conducted to determine the prevalence and infection levels of the microsporidia fungi Nosema apis and/or Nosema ceranae in honey bee colonies of two Canadian provinces. Three surveys were conducted in the springs of 2008, 2010 and 2012 and PCR identification of Nosema species were performed in samples from 169 and 181 Ontario colonies and from 76 Alberta colonies that tested positive to Nosema spp. Infection levels of positive colonies were determined by microscopy and analyzed by Nosema spp. Results showed that N. ceranae was the dominant species in all three surveys (prevalence range of 41-91 vs. 4-34 % for N. apis), whereas mixed infections were less frequent than single infections (5-25 %). Infection levels of colonies parasitized by N. ceranae were three to five times higher than those of colonies parasitized by N. apis in the three surveys whereas mixed infections showed the highest spore counts. This is the first field study demonstrating significantly higher infection levels in colonies parasitized with either N. ceranae only or with both, N. ceranae and N. apis, than in colonies parasitized with N. apis only. Taken together, these results suggest that N. ceranae may be more virulent and better adapted than N. apis in cold climates such as Canadian environments.

Bumblebees are highly valued for their pollination services in natural ecosystems as well as for agricultural crops. These precious pollinators are known to be declining worldwide, and one major factor contributing to this decline are infections by parasites. Knowledge about parasites in wild bumblebee populations is thus of paramount importance for conservation purposes. We here report the geographical distribution of Crithidia and Nosema, two common parasites of bumblebees, in a yet poorly investigated country: Mexico. Based on sequence divergence of the Cytochrome b and Glycosomal glyceraldehyde phosphate deshydrogenase (gGPDAH) genes, we discovered the presence of a new Crithidia species, which is mainly distributed in the southern half of the country. It is placed by Bayesian inference as a sister species to C. bombi. We suggest the name Crithidia mexicana for this newly discovered organism. A population of C. expoeki was encountered concentrated on the flanks of the dormant volcanic mountain, Iztaccihuatl, and microsatellite data showed evidence of a bottleneck in this population. This study is the first to provide a large-scale insight into the health status of endemic bumblebees in Mexico, based on a large sample size (n=3,285 bees examined) over a variety of host species and habitats., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Two microsporidians are known to infect honey bees: Nosema apis and Nosema ceranae. Whereas population genetics data for the latter have been released in the last few years, such information is still missing for N. apis. Here we analyze the patterns of nucleotide polymorphism at three single-copy loci (PTP2, PTP3 and RPB1) in a collection of Apis mellifera isolates from all over the world, naturally infected either with N. apis (N = 22) or N. ceranae (N = 23), to provide new insights into the genetic diversity, demography and evolution of N. apis, as well as to compare them with evidence from N. ceranae. Neutral variation in N. apis and N. ceranae is of the order of 1%. This amount of diversity suggests that there is no substantial differentiation between the genetic content of the two nuclei present in these parasites, and evidence for genetic recombination provides a putative mechanism for the flow of genetic information between chromosomes. The analysis of the frequency spectrum of neutral variants reveals a significant surplus of low frequency variants, particularly in N. ceranae, and suggests that the populations of the two pathogens are not in mutation-drift equilibrium and that they have experienced a population expansion. Most of the variation in both species occurs within honey bee colonies (between 62%-90% of the total genetic variance), although in N. apis there is evidence for differentiation between parasites isolated from distinct A. mellifera lineages (20%-34% of the total variance), specifically between those collected from lineages A and C (or M). This scenario is consistent with a long-term host-parasite relationship and contrasts with the lack of differentiation observed among host-lineages in N. ceranae (< 4% of the variance), which suggests that the spread of this emergent pathogen throughout the A. mellifera worldwide population is a recent event.

Nosema ceranae is a microsporidian pathogen whose infections have been associated with recent global declines in the populations of western honeybees (Apis mellifera). Despite the outstanding economic and ecological threat that N. ceranae may represent for honeybees worldwide, many aspects of its biology, including its mode of reproduction, propagation and ploidy, are either very unclear or unknown. In the present study, we set to gain knowledge in these biological aspects by re-sequencing the genome of eight isolates (i.e. a population of spores isolated from one single beehive) of this species harvested from eight geographically distant beehives, and by investigating their level of polymorphism. Consistent with previous analyses performed using single gene sequences, our analyses uncovered the presence of very high genetic diversity within each isolate, but also very little hive-specific polymorphism. Surprisingly, the nature, location and distribution of this genetic variation suggest that beehives around the globe are infected by a population of N. ceranae cells that may be polyploid (4n or more), and possibly clonal. Lastly, phylogenetic analyses based on genome-wide single-nucleotide polymorphism data extracted from these parasites and mitochondrial sequences from their hosts all failed to support the current geographical structure of our isolates., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Nosema bombi is an obligate intracellular parasite of bumblebees (Hymenoptera, Bombus spp.), which has significant negative effect on individual bumblebees, colony fitness, and development. Recently, several new genetic variants of N. bombi without a defined taxonomic status were identified in natural bumblebee populations from Russia, China, and several European countries, as well as N. ceranae, originally isolated from honey bees, was described in bumblebee species. Thus, it is required to investigate more Nosema variability in bumblebee populations for identifying new genetic Nosema variants. In our study, we used several methods such as total DNA isolation, polymerase chain reaction (PCR) amplification, cloning, sequencing, and comparative and phylogenetic analysis to investigate a prevalence of N. bombi and its diversity in the natural populations of bumblebees across West Siberia. DNA was extracted from intestinal bumblebee tissues. Identification of the parasite was conducted, using PCR with primers specific for the ribosomal RNA gene cluster and methionine aminopeptidase 2 gene of N. bombi followed by sequencing. Seven hundred twenty-seven individual bumblebees belonging to 16 species were tested; 64 specimens revealed presence of the parasite. Prevalence of Nosema bombi infection was different in each region and varied from 4 to 20 %. No infection was found in Bombus agrorum (n = 194) and Bombus equestris (n = 132), both common bumblebees in West Siberia. Three different genetic variants of the same species, N. bombi, were identified. The first variant belonged to N. bombi (AY008373) identified by Fies et al. (J Apicult Res 40:91-96, 2001), second (N. bombi WS2) was identical to the West Siberian variant identified by Szentgyörgyi et al. (Polish Journal of Ecology 59:599-610, 2011), and the last variant, N. bombi WS3, was new. The results led us to suggest that the prevalence of the N. bombi is related to the population structure of bumblebees and distribution of the particular genetic variants of N. bombi.

The queens of eusocial bees, ants and wasps mate only during a very short period early in life and males therefore produce ejaculates consisting of large numbers of high quality sperm. Such extreme selection for high fecundity resulted in males investing minimally into their somatic survival, including their immune system. However, if susceptible males are unable to protect their reproductive tissue from infections, they compromise queen fitness if they transfer pathogens during mating. We used the honey bee Apis mellifera and investigated the course of infection of the sexually transmitted pathogen Nosema apis. We predicted that honey bee males are susceptible but protect their reproductive tissues from infections. We investigated the effects of N. apis infections on the midgut, the accessory glands and the accessory testes and quantified the consequences of infection on male survival and fecundity. We found that N. apis is able to infect males, and as infections progressed, it significantly impacted fertility and survival in older males. Even though we confirm males to be able to minimize N. apis infections of their reproductive tissues, the parasite is present in ejaculates of older males. Consequently N. apis evolved alternative routes to successfully infect ejaculates and get sexually transmitted.

Multiple mating (and insemination) by females with different males, polyandry, is widespread across animals, due to material and/or genetic benefits for females. It reaches particularly high levels in some social insects, in which queens can produce significantly fitter colonies by being polyandrous. It is therefore a paradox that two thirds of eusocial hymenopteran insects appear to be exclusively monandrous, in spite of the fitness benefits that polyandry could provide. One possible cost of polyandry could be sexually transmitted parasites, but evidence for these in social insects is extremely limited. Here we show that two different species of Nosema microsporidian parasites can transmit sexually in the honey bee Apis mellifera. Honey bee males that are infected by the parasite have Nosema spores in their semen, and queens artificially inseminated with either Nosema spores or the semen of Nosema-infected males became infected by the parasite. The emergent and more virulent N. ceranae achieved much higher rates of infection following insemination than did N. apis. The results provide the first quantitative evidence of a sexually transmitted disease (STD) in social insects, indicating that STDs may represent a potential cost of polyandry in social insects.

Nosema ceranae causes a widespread disease that reduces honey bee health but is only thought to infect adult honey bees, not larvae, a critical life stage. We reared honey bee (Apis mellifera) larvae in vitro and provide the first demonstration that N. ceranae can infect larvae and decrease subsequent adult longevity. We exposed three-day-old larvae to a single dose of 40,000 (40K), 10,000 (10K), zero (control), or 40K autoclaved (control) N. ceranae spores in larval food. Spores developed intracellularly in midgut cells at the pre-pupal stage (8 days after egg hatching) of 41% of bees exposed as larvae. We counted the number of N. ceranae spores in dissected bee midguts of pre-pupae and, in a separate group, upon adult death. Pre-pupae exposed to the 10K or 40K spore treatments as larvae had significantly elevated spore counts as compared to controls. Adults exposed as larvae had significantly elevated spore counts as compared to controls. Larval spore exposure decreased longevity: a 40K treatment decreased the age by which 75% of adult bees died by 28%. Unexpectedly, the low dose (10K) led to significantly greater infection (1.3 fold more spores and 1.5 fold more infected bees) than the high dose (40K) upon adult death. Differential immune activation may be involved if the higher dose triggered a stronger larval immune response that resulted in fewer adult spores but imposed a cost, reducing lifespan. The impact of N. ceranae on honey bee larval development and the larvae of naturally infected colonies therefore deserve further study.

The microsporidian Nosema ceranae is an emergent pathogen that threatens the health of honeybees and other pollinators all over the world. Its recent rapid spread across a wide variety of host species and environments demonstrated an enhanced ability of adaptation, which seems to contradict the lack of evidence for genetic recombination and the absence of a sexual stage in its life cycle. Here we retrieved fresh data of the patterns of genetic variation at the PTP2 locus in naturally infected Apis mellifera colonies, by means of single genome amplification. This technique, designed to prevent the formation of chimeric haplotypes during polymerase chain reaction (PCR), provides more reliable estimates of the diversity levels and haplotype structure than standard PCR-cloning methods. Our results are consistent with low but significant rates of recombination in the history of the haplotypes detected: estimates of the population recombination rate are of the order of 30 and support recent evidence for unexpectedly high levels of variation of the parasites within honeybee colonies. These observations suggest the existence of a diploid stage at some point in the life cycle of this parasite and are relevant for our understanding of the dynamics of its expanding population., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Nosema ceranae has been found infecting Apismellifera colonies with increasing frequency and it now represents a major threat to the health and long-term survival of these honeybees worldwide. However, so far little is known about the population genetics of this parasite. Here, we describe the patterns of genetic variation at three genomic loci in a collection of isolates from all over the world. Our main findings are: (i) the levels of genetic polymorphism (πS≈1%) do not vary significantly across its distribution range, (ii) there is substantial evidence for recombination among haplotypes, (iii) the best part of the observed genetic variance corresponds to differences within bee colonies (up to 88% of the total variance), (iv) parasites collected from Asian honeybees (Apis cerana and Apis florea) display significant differentiation from those obtained from Apismellifera (8-16% of the total variance, p<0.01) and (v) there is a significant excess of low frequency variants over neutral expectations among samples obtained from A. mellifera, but not from Asian honeybees. Overall these results are consistent with a recent colonization and rapid expansion of N. ceranae throughout A. mellifera colonies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

About 20% of bumble bee species are in decline in North America, and the microsporidian pathogen, Nosema bombi, has been correlated with these declines. We conducted a comprehensive survey of N. bombi infections in the bumble bee communities throughout the flight season along an elevation gradient in Northern Arizona. Focusing on two species, Bombus (Pyrobombus) huntii and Bombus (Pyrobombus) centralis, we used a combination of PCR and microscopy to distinguish between sporulating and non/low, sporulating N. bombi infections. Surprisingly high levels of PCR-positive infections with no detectable spore loads were found in B. huntii (31-63%) and B. centralis (56.5-66.5%), while the prevalence of sporulating infections was low (3.0-11.8% and 0-12.9% respectively). We determined the prevalence of sporulating N. bombi infection in six other co-occurring, but rarer, bumble bee species (0-62.5%,), but did not test them using PCR. The prevalence of sporulating N. bombi infections in B. (Bombias) nevadensis was significantly higher than in either B. huntii or B. centralis (29%). The declining bumble bee, Bombus sensu strico occidentalis, had the highest prevalence of sporulating N. bombi infections (62.5%), but we purposely captured very few B. occidentalis because of its declining status. PCR was a more sensitive measure of N. bombi prevalence and revealed that wild bumble bees have a much higher prevalence of N. bombi than has previously been recognized. Microscopy and PCR together provide complementary, not redundant, information that deepens our understanding of the dynamic interactions between N. bombi and their bumble bee hosts., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Here we present a holistic screening of collapsing colonies from three professional apiaries in Spain. Colonies with typical honey bee depopulation symptoms were selected for multiple possible factors to reveal the causes of collapse., Results: Omnipresent were Nosema ceranae and Lake Sinai Virus. Moderate prevalences were found for Black Queen Cell Virus and trypanosomatids, whereas Deformed Wing Virus, Aphid Lethal Paralysis Virus strain Brookings and neogregarines were rarely detected. Other viruses, Nosema apis, Acarapis woodi and Varroa destructor were not detected. Palinologic study of pollen demonstrated that all colonies were foraging on wild vegetation. Consequently, the pesticide residue analysis was negative for neonicotinoids. The genetic analysis of trypanosomatids GAPDH gene, showed that there is a large genetic distance between Crithidia mellificae ATCC30254, an authenticated cell strain since 1974, and the rest of the presumed C. mellificae sequences obtained in our study or published. This means that the latter group corresponds to a highly differentiated taxon that should be renamed accordingly., Conclusion: The results of this study demonstrate that the drivers of colony collapse may differ between geographic regions with different environmental conditions, or with different beekeeping and agricultural practices. The role of other pathogens in colony collapse has to bee studied in future, especially trypanosomatids and neogregarines. Beside their pathological effect on honey bees, classification and taxonomy of these protozoan parasites should also be clarified.

Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed at a constant temperature of 60 °C using two sets of six species-specific primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 10(3) -fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Infection of honeybees by the microsporidian Nosema ceranae is considered to be one of the factors underlying the increased colony losses and decreased honey production seen in recent years. However, these effects appear to differ in function of the climatic zone, the distinct beekeeping practices and the honeybee species employed. Here, we compared the response of Apis mellifera iberiensis worker bees to experimental infection with field isolates of N. ceranae from an Oceanic climate zone in Northern Europe (Netherlands) and from a Mediterranean region of Southern Europe (Spain). We found a notable but non-significant trend (P = 0.097) towards higher honeybee survival for bees infected with N. ceranae from the Netherlands, although no differences were found between the two isolates in terms of anatomopathological lesions in infected ventricular cells or the morphology of the mature and immature stages of the parasite. In addition, the population genetic survey of the N. ceranae PTP3 locus revealed high levels of genetic diversity within each isolate, evidence for meiotic recombination, and no signs of differentiation between the Dutch and Spanish populations. A cross-infection study is needed to further explore the differences in virulence observed between the two N. ceranae populations in field conditions., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The Palaearctic Bombus ruderatus (in 1982/1983) and Bombus terrestris (1998) have both been introduced into South America (Chile) for pollination purposes. We here report on the results of sampling campaigns in 2004, and 2010-2012 showing that both species have established and massively expanded their range. Bombus terrestris, in particular, has spread by some 200 km year(-1) and had reached the Atlantic coast in Argentina by the end of 2011. Both species, and especially B. terrestris, are infected by protozoan parasites that seem to spread along with the imported hosts and spillover to native species. Genetic analyses by polymorphic microsatellite loci suggest that the host population of B. terrestris is genetically diverse, as expected from a large invading founder population, and structured through isolation by distance. Genetically, the populations of the trypanosomatid parasite, Crithidia bombi, sampled in 2004 are less diverse, and distinct from the ones sampled later. Current C. bombi populations are highly heterozygous and also structured through isolation by distance correlating with the genetic distances of B. terrestris, suggesting the latter's expansion to be a main structuring factor for the parasite. Remarkably, wherever B. terrestris spreads, the native Bombus dahlbomii disappears although the reasons remain unclear. Our ecological and genetic data suggest a major invasion event that is currently unfolding in southern South America with disastrous consequences for the native bumblebee species., (© 2013 The Authors. Journal of Animal Ecology © 2013 British Ecological Society.)

Microsporidiosis caused by Nosema species is one of the factors threatening the health of the honeybee (Apis mellifera), which is an essential element in agriculture mainly due to its pollination function. The dispersion of this pathogen may be influenced by many factors, including various aspects of beekeeping management such as introduction of queens with different origin. Herein we study the relation of the presence and distribution of Nosema spp. and the replacement of queens in honeybee populations settled on the Atlantic Canary Islands. While Nosema apis has not been detected, an increase of the presence and distribution of Nosema ceranae during the last decade has been observed in parallel with a higher frequency of foreign queens. On the other hand, a reduction of the number of N. ceranae positive colonies was observed on those islands with continued replacement of queens. We suggest that such replacement could help maintaining low rates of Nosema infection, but healthy queens native to these islands should be used in order to conserve local honeybee diversity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Nosema ceranae is a widespread honeybee parasite, considered to be one of the pathogens involved in the colony losses phenomenon. To date, little is known about its intraspecific genetic variability. The few studies on N. ceranae variation have focused on the subunits of ribosomal DNA, which are not ideal for this purpose and have limited resolution. Here we characterized three single copy loci (Actin, Hsp70 and RPB1) in three N. ceranae isolates from Hungary and Hawaii. Our results provide evidence of unexpectedly high levels of intraspecific polymorphism, the coexistence of a wide variety of haplotypes within each bee colony, and the occurrence of genetic recombination in RPB1. Most haplotypes are not shared across isolates and derive from a few frequent haplotypes by a reduced number of singletons (mutations that appear usually just once in the sample), which suggest that they have a fairly recent origin. Overall, our data indicate that this pathogen has experienced a recent population expansion. The presence of multiple haplotypes within individual isolates could be explained by the existence of different strains of N. ceranae infecting honeybee colonies in the field which complicates, and must not be overlooked, further analysis of host-parasite interactions.

To date, few organisms have been shown to possess variable ribosomal RNA, otherwise considered a classic example of uniformity by concerted evolution. The polymorphism for the 16S rRNA in Nosema ceranae analysed here is striking as Microsporidia are intracellular parasites which have suffered a strong reduction in their genomes and cellular organization. Moreover, N. ceranae infects the honeybee Apis mellifera, and has been associated with the colony-loss phenomenon during the last decade. The variants of 16S rRNA include single nucleotide substitutions, one base insertion-deletion, plus a tetranucleotide indel. We show that different gene variants are expressed. The polymorphic sites tend to be located in particular regions of the rRNA molecule, and the comparison to the Escherichia coli 16S rRNA secondary structure indicates that most variations probably do not preclude ribosomal activity. The fact that the polymorphisms in such a minimal organism as N. ceranae are maintained in samples collected worldwide suggest that the existence of differently expressed rRNA may play an adaptive role in the microsporidian., (© 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chronic bee paralysis virus (CBPV) is an important viral disease of adult bees which induces significant losses in honey bee colonies. Despite comprehensive research, only limited data is available from experimental infection for this virus. In the present study winter worker bees were experimentally infected in three different experiments. Bees were first inoculated per os (p/o) or per cuticle (p/c) with CBPV field strain M92/2010 in order to evaluate the virus replication in individual bees. In addition, potential synergistic effects of co-infection with CBPV and Nosema ceranae (N. ceranae) on bees were investigated. In total 558 individual bees were inoculated in small cages and data were analyzed using quantitative real time RT-PCR (RT-qPCR). Our results revealed successful replication of CBPV after p/o inoculation, while it was less effective when bees were inoculated p/c. Dead bees harbored about 1,000 times higher copy numbers of the virus than live bees. Co-infection of workers with CBPV and N. ceranae using either method of virus inoculation (p/c or p/o) showed increased replication ability for CBPV. In the third experiment the effect of inoculation on bee mortality was evaluated. The highest level of bee mortality was observed in a group of bees inoculated with CBPV p/o, followed by a group of workers simultaneously inoculated with CBPV and N. ceranae p/o, followed by the group inoculated with CBPV p/c and the group with only N. ceranae p/o. The experimental infection with CBPV showed important differences after p/o or p/c inoculation in winter bees, while simultaneous infection with CBPV and N. ceranae suggesting a synergistic effect after inoculation.

The microsporidian parasite Nosema ceranae is a common pathogen of the Western honeybee (Apis mellifera) whose variable virulence could be related to its genetic polymorphism and/or its polyphenism responding to environmental cues. Since the genotyping of N. ceranae based on unique marker sequences had been unsuccessful, we tested whether a multilocus approach, assessing the diversity of ten genetic markers – encoding nine proteins and the small ribosomal RNA subunit – allowed the discrimination between N. ceranae variants isolated from single A. mellifera individuals in four distant locations. High nucleotide diversity and allele content were observed for all genes. Most importantly, the diversity was mainly present within parasite populations isolated from single honeybee individuals. In contrast the absence of isolate differentiation precluded any taxa discrimination, even through a multilocus approach, but suggested that similar populations of parasites seem to infect honeybees in distant locations. As statistical evolutionary analyses showed that the allele frequency is under selective pressure, we discuss the origin and consequences of N. ceranae heterozygosity in a single host and lack of population divergence in the context of the parasite natural and evolutionary history.

The loss of Apis mellifera L. colonies in recent years has, in many regions of the world, been alarmingly high. No single cause has been identified for these losses, but the interactions between several factors (mostly pathogens and parasites) have been held responsible. Work in the Americas on honeybees originating mainly from South Africa indicates that Africanised honeybees are less affected by the interplay of pathogens and parasites. However, little is known about the health status of South African honeybees (A. m. scutellata and A. m. capensis) in relation to pathogens and parasites. We therefore compared the seasonal prevalence of honeybee pathogens (viruses, bacteria, fungi) and parasites (mites, bee lice, wax moth, small hive beetles, A. m. capensis social parasites) between sedentary and migratory A. m. scutellata apiaries situated in the Gauteng region of South Africa. No significant differences were found in the prevalence of pathogens and parasites between sedentary and migratory apiaries. Three (Black queen cell virus, Varroa destructor virus 1 and Israeli acute paralysis virus) of the eight viruses screened were detected, a remarkable difference compared to European honeybees. Even though no bacterial pathogens were detected, Nosema apis and Chalkbrood were confirmed. All of the honeybee parasites were found in the majority of the apiaries with the most common parasite being the Varroa mite. In spite of hosting few pathogens, yet most parasites, A. m. scutellata colonies appeared to be healthy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The microsporidia parasite Nosema contributes to the steep global decline of honey bees that are critical pollinators of food crops. There are two species of Nosema that have been found to infect honey bees, Nosema apis and N. ceranae. Genome sequencing of N. apis and comparative genome analysis with N. ceranae, a fully sequenced microsporidia species, reveal novel insights into host-parasite interactions underlying the parasite infections., Results: We applied the whole-genome shotgun sequencing approach to sequence and assemble the genome of N. apis which has an estimated size of 8.5 Mbp. We predicted 2,771 protein- coding genes and predicted the function of each putative protein using the Gene Ontology. The comparative genomic analysis led to identification of 1,356 orthologs that are conserved between the two Nosema species and genes that are unique characteristics of the individual species, thereby providing a list of virulence factors and new genetic tools for studying host-parasite interactions. We also identified a highly abundant motif in the upstream promoter regions of N. apis genes. This motif is also conserved in N. ceranae and other microsporidia species and likely plays a role in gene regulation across the microsporidia., Conclusions: The availability of the N. apis genome sequence is a significant addition to the rapidly expanding body of microsprodian genomic data which has been improving our understanding of eukaryotic genome diversity and evolution in a broad sense. The predicted virulent genes and transcriptional regulatory elements are potential targets for innovative therapeutics to break down the life cycle of the parasite.

The intestinal honey bee parasite Nosema ceranae (Microsporidia) is at the root of colony losses in some regions while in others its presence causes no direct mortality. This is the case for Spain and France, respectively. It is hypothesized that differences in honey bee responses to N. ceranae infection could be due to the degree of virulence of N. ceranae strains from different geographic origins. To test this hypothesis, we first performed a study to compare the genetic variability of an rDNA fragment that could reveal differences between two N. ceranae isolates, one from Spain and one from France. Then we compared the infection capacity of both isolates in Apis mellifera iberiensis, based on the anatomopathological lesions due to N. ceranae development in the honey bee midgut, N. ceranae spore-load in the midgut and the honey bee survival rate. Our results suggest that there is no specific genetic background of the two N. ceranae isolates, from Spain or France, used in this study. These results agree with the infection development, honey bee survival and spore-loads that were similar between honey bees infected with both N. ceranae isolates. Probably, differences in honey bee response to infection are more related to the degree of tolerance of honey bee subspecies or local hybrids to N. ceranae, or experimental conditions in the case of laboratory trials, than to differences between N. ceranae isolates. Further studies should be done to estimate the contribution of each of these factors on the response of the honey bees to infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The role of protozoan parasites in honey bee health and distribution in the world is not well understood. Therefore, we carried out a molecular survey for the presence of Crithidia mellificae and Apicystis bombi in the colonies of both non-native Apis mellifera and native Apis cerana japonica in Japan. We found that A. mellifera, but not A. c. japonica, colonies are parasitized with C. mellificae and A. bombi. Their absence in A. c. japonica colonies indicates that A. mellifera is their native host. Nevertheless, the prevalence in A. mellifera colonies is low compared with other pathogens such as viruses and Nosema microsporidia. Japanese C. mellificae isolates share well-conserved nuclear-encoded gene sequences with Swiss and US isolates. We have found two Japanese haplotypes (A and B) with two nucleotide differences in the kinetoplast-encoded cytochrome b sequence. The haplotype A is identical to Swiss isolate. These results demonstrate that C. mellificae and A. bombi distribute in Asia, Oceania, Europe, and South and North Americas., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

In a nationwide Swedish survey, 967 honey bee colonies from 521 beekeepers were sampled in the spring of 2007 and the samples assayed for Nosema spp. infections. Of the 319 positive samples, only 32 samples contained a proportion of N. ceranae DNA in mixed infections with both Nosema spp. above the cut-off point chosen for comparisons of 1%. Only one pure N. ceranae infection was found, with the rest 284 infected samples assayed being pure N. apis infections. In 2009 and 2011, beekeepers or bee inspectors providing N. ceranae mixed positive bee samples in 2007 were again asked to submit samples (2009, n = 96; 2011, n = 83). No trend of an increased proportion of N. ceranae-infected samples could be found. The proportion of N. ceranae DNA in samples with mixed infection did not increase between 2007 and 2011. It is concluded that N. apis is still the dominating Microsporidia infection in honey bees in Sweden and that there is no tendency for one species replacing the other., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)