Development and evaluation of a core genome multilocus sequence typing scheme for Paenibacillus larvae, the deadly American foulbrood pathogen of honeybees.

Paenibacillus larvae is the causative agent of the fatal American foulbrood disease in honeybees (Apis mellifera). Strain identification is vital for preventing the spread of the disease. To date, the most accessible and robust scheme to identify strains is the multilocus sequence typing (MLST) method. However, this approach has limited resolution, especially for epidemiological studies. As the cost of whole-genome sequencing has decreased and as it becomes increasingly available to most laboratories, an extended MLST based on the core genome (cgMLST) presents a valuable tool for high-resolution investigations. In this study, we present a standardized, robust cgMLST scheme for P. larvae typing using whole-genome sequencing. A total of 333 genomes were used to identify, validate and evaluate 2419 core genes. The cgMLST allowed fine-scale differentiation between samples that had the same profile using traditional MLST and allowed for the characterization of strains impossible by MLST. The scheme was successfully used to trace a localized Swedish outbreak, where a cluster of 38 isolates was linked to a country-wide beekeeping operation. cgMLST greatly enhances the power of a traditional typing scheme, while preserving the same stability and standardization for sharing results and methods across different laboratories.

Interactions Between Thiamethoxam and Deformed Wing Virus Can Drastically Impair Flight Behavior of Honey Bees.

Exposure to multiple stress factors is believed to contribute to honey bee colony decline. However, little is known about how co-exposure to stress factors can alter the survival and behavior of free-living honey bees in colony conditions. We therefore studied the potential interaction between a neonicotinoid pesticide, thiamethoxam, and a highly prevalent honey bee pathogen, Deformed wing virus (DWV). For this purpose, tagged bees were exposed to DWV by feeding or injection, and/or to field-relevant doses of thiamethoxam, then left in colonies equipped with optical bee counters to monitor flight activity. DWV loads and the expression of immune genes were quantified. A reduction in vitellogenin expression level was observed in DWV-injected bees and was associated with precocious onset of foraging. Combined exposure to DWV and thiamethoxam did not result in higher DWV loads compared to bees only exposed to DWV, but induced precocious foraging, increased the risk of not returning to the hive after the first flight, and decreased survival when compared to single stress exposures. We therefore provided the first evidence for deleterious interactions between DWV and thiamethoxam in natural conditions.

Influence of chronic exposure to thiamethoxam and chronic bee paralysis virus on winter honey bees.

Co-exposure to pesticides and viruses is likely to occur in honey bee colonies. Pesticides can be present in pollen, nectar, and persist in stored food (honey and bee bread), and viruses can be highly prevalent in honey bee colonies. Therefore, the present study describes the influence of chronic co-exposure to thiamethoxam and Chronic bee paralysis virus (CBPV) on bee survival, virus loads, expression level of immune and detoxication genes, and pesticide metabolism Experiments were performed on honey bees collected from a winter apiary with reduced viral contaminations. No synergistic effect of co-exposure was observed on bee survival, nor on the ability of bees to metabolise the pesticide into clothianidin. However, we found that co-exposure caused an increase in CBPV loads that reached the viral levels usually found in overt infections. The effect of co-exposure on CBPV replication was associated with down-regulation of vitellogenin and dorsal-1a gene transcription. Nevertheless, the observed effects might be different to those occurring in spring or summer bees, which are more likelyco-exposed to thiamethoxam and CBPV and exhibit a different physiology.

Validation of quantitative real-time RT-PCR assays for the detection of six honeybee viruses.

Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Chronic bee paralysis virus (CBPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Varroa destructor virus 1 (VDV1) are the six main honeybee viruses reported in Europe. We assessed the accuracy (trueness and precision) of reverse transcriptase quantitative TaqMan® PCR methods (RT-qPCR) for quantifying ABPV, BQCV, DWV, VDV1 and SBV loads. Once the systematic bias in quantitative results had been corrected (overestimation in ABPV and BQCV quantification and underestimation in that of SBV and VDV1), measurements were taken to determine the viral load ranges for which quantification uncertainty was below ± 1 log10 equivalent of genome copies per bee (hereafter reported as genome copies/bee). The accuracy range of RT-qPCR was found to be between 6.4 and 10.4 log10 genome copies/bee for ABPV, between 3.0 and 10.0 log10 genome copies/bee for BQCV, between 2.4 and 10.4 log10 genome copies/bee for DWV and between 3.4 and 10.4 log10 genome copies/bee for SBV. Outside these ranges, the results' uncertainty is higher. VDV1 RT-qPCR accuracy was outside accuracy limits for all viral loads. Using these RT-qPCR methods, we quantified viral loads in naturally-infected honeybees. The viral load distribution and clinical signs reported with the honeybee samples allowed us to define a threshold that could be used to differentiate between covert and overt infections. These methods will be useful in diagnosing the main viral infections impairing honeybee health.

Complete Mitochondrial Genome Sequence of Aethina tumida (Coleoptera: Nitidulidae), a Beekeeping Pest.

We report here the full mitochondrial genome sequence of Aethina tumida, a Nitidulidae species beetle, that is a pest of bee hives. The obtained sequence is 16,576 bp in length and contains 13 protein-coding genes, 2 rRNA genes, and 22 tRNAs.

Trueness and precision of the real-time RT-PCR method for quantifying the chronic bee paralysis virus genome in bee homogenates evaluated by a comparative inter-laboratory study.

The Chronic bee paralysis virus (CBPV) is the aetiological agent of chronic bee paralysis, a contagious disease associated with nervous disorders in adult honeybees leading to massive mortalities in front of the hives. Some of the clinical signs frequently reported, such as trembling, may be confused with intoxication syndromes. Therefore, laboratory diagnosis using real-time PCR to quantify CBPV loads is used to confirm disease. Clinical signs of chronic paralysis are usually associated with viral loads higher than 108 copies of CBPV genome copies per bee (8 log10 CBPV/bee). This threshold is used by the European Union Reference Laboratory for Bee Health to diagnose the disease. In 2015, the accuracy of measurements of three CBPV loads (5, 8 and 9 log10 CBPV/bee) was assessed through an inter-laboratory study. Twenty-one participants, including 16 European National Reference Laboratories, received 13 homogenates of CBPV-infected bees adjusted to the three loads. Participants were requested to use the method usually employed for routine diagnosis. The quantitative results (n=270) were analysed according to international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). The standard deviations of measurement reproducibility (SR) were 0.83, 1.06 and 1.16 at viral loads 5, 8 and 9 log10 CBPV/bee, respectively. The inter-laboratory confidence of viral quantification (+/- 1.96SR) at the diagnostic threshold (8 log10 CBPV/bee) was+/- 2.08 log10 CBPV/bee. These results highlight the need to take into account the confidence of measurements in epidemiological studies using results from different laboratories. Considering this confidence, viral loads over 6 log10 CBPV/bee may be considered to indicate probable cases of chronic paralysis.

A pan-European epidemiological study reveals honey bee colony survival depends on beekeeper education and disease control.

Reports of honey bee population decline has spurred many national efforts to understand the extent of the problem and to identify causative or associated factors. However, our collective understanding of the factors has been hampered by a lack of joined up trans-national effort. Moreover, the impacts of beekeeper knowledge and beekeeping management practices have often been overlooked, despite honey bees being a managed pollinator. Here, we established a standardised active monitoring network for 5 798 apiaries over two consecutive years to quantify honey bee colony mortality across 17 European countries. Our data demonstrate that overwinter losses ranged between 2% and 32%, and that high summer losses were likely to follow high winter losses. Multivariate Poisson regression models revealed that hobbyist beekeepers with small apiaries and little experience in beekeeping had double the winter mortality rate when compared to professional beekeepers. Furthermore, honey bees kept by professional beekeepers never showed signs of disease, unlike apiaries from hobbyist beekeepers that had symptoms of bacterial infection and heavy Varroa infestation. Our data highlight beekeeper background and apicultural practices as major drivers of honey bee colony losses. The benefits of conducting trans-national monitoring schemes and improving beekeeper training are discussed.

RNA 1 and RNA 2 Genomic Segments of Chronic Bee Paralysis Virus Are Infectious and Induce Chronic Bee Paralysis Disease.

Chronic bee paralysis virus (CBPV) causes an infectious and contagious disease of adult honeybees. Its segmented genome is composed of two major positive single-stranded RNAs, RNA 1 (3,674 nt) and RNA 2 (2,305 nt). Three minor RNAs (about 1,000 nt each) have been described earlier but they were not detected by sequencing of CBPV genome. In this study, the results of in vivo inoculation of the two purified CBPV major RNAs are presented and demonstrate that RNA 1 and RNA 2 are infectious. Honeybees inoculated with 10(9) RNA copies per bee developed paralysis symptoms within 6 days after inoculation. The number of CBPV RNA copies increased significantly throughout the infection. Moreover, the negative strand of CBPV RNA was detected by RT-PCR, and CBPV particles were visualized by electronic microscopy in inoculated honeybees. Taken together, these results show that CBPV RNA 1 and CBPV RNA 2 segments can induce virus replication and produce CBPV virus particles. Therefore, the three minor RNAs described in early studies are not essential for virus replication. These data are crucial for the development of a reverse genetic system for CBPV.

Characterisation of Structural Proteins from Chronic Bee Paralysis Virus (CBPV) Using Mass Spectrometry.

Chronic bee paralysis virus (CBPV) is the etiological agent of chronic paralysis, an infectious and contagious disease in adult honeybees. CBPV is a positive single-stranded RNA virus which contains two major viral RNA fragments. RNA 1 (3674 nt) and RNA 2 (2305 nt) encode three and four putative open reading frames (ORFs), respectively. RNA 1 is thought to encode the viral RNA-dependent RNA polymerase (RdRp) since the amino acid sequence derived from ORF 3 shares similarities with the RdRP of families Nodaviridae and Tombusviridae. The genomic organization of CBPV and in silico analyses have suggested that RNA 1 encodes non-structural proteins, while RNA 2 encodes structural proteins, which are probably encoded by ORFs 2 and 3. In this study, purified CBPV particles were used to characterize virion proteins by mass spectrometry. Several polypeptides corresponding to proteins encoded by ORF 2 and 3 on RNA 2 were detected. Their role in the formation of the viral capsid is discussed.

Demographics of the European apicultural industry.

Over the last few years, many European and North American countries have reported a high rate of disorders (mortality, dwindling and disappearance) affecting honeybee colonies (Apis mellifera). Although beekeeping has become an increasingly professional activity in recent years, the beekeeping industry remains poorly documented in Europe. The European Union Reference Laboratory for Honeybee Health sent a detailed questionnaire to each Member State, in addition to Kosovo and Norway, to determine the demographics and state of their beekeeping industries. Based on data supplied by the National Reference Laboratory for honeybee diseases in each European country, a European database was created to describe the beekeeping industry including the number and types of beekeepers, operation size, industry production, and health (notifiable diseases, mortalities). The total number of beekeepers in Europe was estimated at 620,000. European honey production was evaluated at around 220,000 tons in 2010. The price of honey varied from 1.5 to 40 €/kg depending on the country and on the distribution network. The estimated colony winter mortality varied from 7 to 28% depending on the country and the origin of the data (institutional survey or beekeeping associations). This survey documents the high heterogeneity of the apicultural industry within the European Union. The high proportion of non-professional beekeepers and the small mean number of colonies per beekeeper were the only common characteristics at European level. The tremendous variation in European apicultural industries has implication for any comprehensive epidemiological or economic analysis of the industry. This variability needs to be taken into account for such analysis as well as for future policy development. The industry would be served if beekeeping registration was uniformly implemented across member states. Better information on the package bee and queen production would help in understanding the ability of the industry to replace lost honey bee stocks.