Since the last decade, unusually high honey bee colony losses have been reported mainly in North-America and Europe. Here, we report on a comprehensive bee pathogen screening in Belgium covering 363 bee colonies that were screened for 18 known disease-causing pathogens and correlate their incidence in summer with subsequent winter mortality. Our analyses demonstrate that, in addition to Varroa destructor, the presence of the trypanosomatid parasite Crithidia mellificae and the microsporidian parasite Nosema ceranae in summer are also predictive markers of winter mortality, with a negative synergy being observed between the two in terms of their effects on colony mortality. Furthermore, we document the first occurrence of a parasitizing phorid fly in Europe, identify a new fourth strain of Lake Sinai Virus (LSV), and confirm the presence of other little reported pathogens such as Apicystis bombi, Aphid Lethal Paralysis Virus (ALPV), Spiroplasma apis, Spiroplasma melliferum and Varroa destructor Macula-like Virus (VdMLV). Finally, we provide evidence that ALPV and VdMLV replicate in honey bees and show that viruses of the LSV complex and Black Queen Cell Virus tend to non-randomly co-occur together. We also noticed a significant correlation between the number of pathogen species and colony losses. Overall, our results contribute significantly to our understanding of honey bee diseases and the likely causes of their current decline in Europe.

Trypanosomatids are increasingly recognized as prevalent in European honey bees (Apis mellifera) and by default are attributed to one recognized species, Crithidia mellificae Langridge and McGhee, 1967. We provide reference genetic and ultrastructural data for type isolates of C. mellificae (ATCC 30254 and 30862) in comparison with two recent isolates from A. mellifera (BRL and SF). Phylogenetics unambiguously identify strains BRL/SF as a novel taxonomic unit distinct from C. mellificae strains 30254/30862 and assign all four strains as lineages of a novel clade within the subfamily Leishmaniinae. In vivo analyses show strains BRL/SF preferably colonize the hindgut, lining the lumen as adherent spheroids in a manner identical to previous descriptions from C. mellificae. Microscopy images show motile forms of C. mellificae are distinct from strains BRL/SF. We propose the binomial Lotmaria passim n. gen., n. sp. for this previously undescribed taxon. Analyses of new and previously accessioned genetic data show C. mellificae is still extant in bee populations, however, L. passim n. gen., n. sp. is currently the predominant trypanosomatid in A. mellifera globally. Our findings require that previous reports of C. mellificae be reconsidered and that subsequent trypanosomatid species designations from Hymenoptera provide genetic support.

Solitary bees and honey bees from a neighbouring apiary were screened for a broad set of putative pathogens including protists, fungi, spiroplasmas and viruses. Most sampled bees appeared to be infected with multiple parasites. Interestingly, viruses exclusively known from honey bees such as Apis mellifera Filamentous Virus and Varroa destructor Macula-like Virus were also discovered in solitary bees. A microsporidium found in Andrena vaga showed most resemblance to Nosema thomsoni. Our results suggest that bee hives represent a putative source of pathogens for other pollinators. Similarly, solitary bees may act as a reservoir of honey bee pathogens.

BackgroundHere 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.ResultsOmnipresent 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-649) contains supplementary material, which is available to authorized users.

The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called “BeeDoctor”, was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. “BeeDoctor” is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. “BeeDoctor” was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the “BeeDoctor”, virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies.

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In a Belgian pilot study honey bee wax combs from ten hives were analyzed on the presence of almost 300 organochlorine and organophosphorous compounds by LC–MS/MS and GC–MS/MS. Traces of 18 pesticides were found and not a single sample was free of residues. The number of residues found per sample ranged from 3 to 13, and the pesticides found could be categorized as (1) pesticides for solely apicultural (veterinary) application, (2) pesticides for solely agricultural (crop protection) application, (3) pesticides for mixed agricultural and apicultural (veterinary) application. The frequencies and quantities of some environmental pollutants bear us high concerns. Most alarming was the detection of lindane (gamma-HCH) and dichlorodiphenyltrichloroethane (including its breakdown product dichlorodiphenyldichloroethylene), two insecticides that are banned in Europe. The present comprehensive residue analysis, however, also reveals residues of pesticides never found in beeswax before, i.e. DEET, propargite and bromophos.