Bacterial community associated with worker honeybees (Apis mellifera) affected by European foulbrood.

BACKGROUND: Melissococcus plutonius is an entomopathogenic bacterium that causes European foulbrood (EFB), a honeybee (Apis mellifera L.) disease that necessitates quarantine in some countries. In Czechia, positive evidence of EFB was absent for almost 40 years, until an outbreak in the Krkonose Mountains National Park in 2015. This occurrence of EFB gave us the opportunity to study the epizootiology of EFB by focusing on the microbiome of honeybee workers, which act as vectors of honeybee diseases within and between colonies. METHODS: The study included worker bees collected from brood combs of colonies (i) with no signs of EFB (EFB0), (ii) without clinical symptoms but located at an apiary showing clinical signs of EFB (EFB1), and (iii) with clinical symptoms of EFB (EFB2). In total, 49 samples from 27 honeybee colonies were included in the dataset evaluated in this study. Each biological sample consisted of 10 surface-sterilized worker bees processed for DNA extraction. All subjects were analyzed using conventional PCR and by metabarcoding analysis based on the 16S rRNA gene V1-V3 region, as performed through Illumina MiSeq amplicon sequencing. RESULTS: The bees from EFB2 colonies with clinical symptoms exhibited a 75-fold-higher incidence of M. plutonius than those from EFB1 asymptomatic colonies. Melissococcus plutonius was identified in all EFB1 colonies as well as in some of the control colonies. The proportions of Fructobacillus fructosus, Lactobacillus kunkeei, Gilliamella apicola, Frischella perrara, and Bifidobacterium coryneforme were higher in EFB2 than in EFB1, whereas Lactobacillus mellis was significantly higher in EFB2 than in EFB0. Snodgrassella alvi and L. melliventris, L. helsingborgensis and, L. kullabergensis exhibited higher proportion in EFB1 than in EFB2 and EFB0. The occurrence of Bartonella apis and Commensalibacter intestini were higher in EFB0 than in EFB2 and EFB1. Enterococcus faecalis incidence was highest in EFB2. CONCLUSIONS: High-throughput Illumina sequencing permitted a semi-quantitative analysis of the presence of M. plutonius within the honeybee worker microbiome. The results of this study indicate that worker bees from EFB-diseased colonies are capable of transmitting M. plutonius due to the greatly increased incidence of the pathogen. The presence of M. plutonius sequences in control colonies supports the hypothesis that this pathogen exists in an enzootic state. The bacterial groups synergic to both the colonies with clinical signs of EFB and the EFB-asymptomatic colonies could be candidates for probiotics. This study confirms that E. faecalis is a secondary invader to M. plutonius; however, other putative secondary invaders were not identified in this study.

Honeybee (Apis mellifera)-associated bacterial community affected by American foulbrood: detection of Paenibacillus larvae via microbiome analysis.

Honeybee (Apis mellifera L.) workers act as passive vectors of Paenibacillus larvae spores, which cause the quarantine disease American foulbrood (AFB). We assessed the relative proportions of P. larvae within the honeybee microbiome using metabarcoding analysis of the 16 S rRNA gene. The microbiome was analyzed in workers outside of the AFB zone (control - AFB0), in workers from asymptomatic colonies in an AFB apiary (AFB1), and in workers from colonies exhibiting clinical AFB symptoms (AFB2). The microbiome was processed for the entire community and for a cut-off microbiome comprising pathogenic/environmental bacteria following the removal of core bacterial sequences; varroosis levels were considered in the statistical analysis. No correlation was observed between AFB status and varroosis level, but AFB influenced the worker bee bacterial community, primarily the pathogenic/environmental bacteria. There was no significant difference in the relative abundance of P. larvae between the AFB1 and AFB0 colonies, but we did observe a 9-fold increase in P. larvae abundance in AFB2 relative to the abundance in AFB1. The relative sequence numbers of Citrobacter freundii and Hafnia alvei were higher in AFB2 and AFB1 than in AFB0, whereas Enterococcus faecalis, Klebsiella oxytoca, Spiroplasma melliferum and Morganella morganii were more abundant in AFB0 and AFB1 than in AFB2.

Variation in honey bee gut microbial diversity affected by ontogenetic stage, age and geographic location.

Social honey bees, Apis mellifera, host a set of distinct microbiota, which is similar across the continents and various honey bee species. Some of these bacteria, such as lactobacilli, have been linked to immunity and defence against pathogens. Pathogen defence is crucial, particularly in larval stages, as many pathogens affect the brood. However, information on larval microbiota is conflicting. Seven developmental stages and drones were sampled from 3 colonies at each of the 4 geographic locations of A. mellifera carnica, and the samples were maintained separately for analysis. We analysed the variation and abundance of important bacterial groups and taxa in the collected bees. Major bacterial groups were evaluated over the entire life of honey bee individuals, where digestive tracts of same aged bees were sampled in the course of time. The results showed that the microbial tract of 6-day-old 5th instar larvae were nearly equally rich in total microbial counts per total digestive tract weight as foraging bees, showing a high percentage of various lactobacilli (Firmicutes) and Gilliamella apicola (Gammaproteobacteria 1). However, during pupation, microbial counts were significantly reduced but recovered quickly by 6 days post-emergence. Between emergence and day 6, imago reached the highest counts of Firmicutes and Gammaproteobacteria, which then gradually declined with bee age. Redundancy analysis conducted using denaturing gradient gel electrophoresis identified bacterial species that were characteristic of each developmental stage. The results suggest that 3-day 4th instar larvae contain low microbial counts that increase 2-fold by day 6 and then decrease during pupation. Microbial succession of the imago begins soon after emergence. We found that bacterial counts do not show only yearly cycles within a colony, but vary on the individual level. Sampling and pooling adult bees or 6th day larvae may lead to high errors and variability, as both of these stages may be undergoing dynamic succession.

In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite.

We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed.

Point mutations in the sodium channel gene conferring tau-fluvalinate resistance in Varroa destructor.

BACKGROUND: Sodium channels (SCs) in mites and insects are target sites for pesticides, including pyrethroids. Point mutations in the SC gene have been reported to change the structural conformation of the protein and its sensitivity to pesticides. To find mutations in the SC gene of the mite Varroa destructor (VmNa), the authors analysed the VmNa gene sequences available in GenBank and prepared specific primers for the amplification of two fragments containing the regions coding for (i) the domain II S4-S6 region (bp 2805-3337) and (ii) the domain III S4-3' terminus region (bp 4737-6500), as determined according to the VmNa cDNA sequence AY259834. RESULTS: Sensitive and resistant mite populations did not differ in the amino acid sequences of the III S4-3' terminus VmNa region. However, differences were found in the IIS4-IIS6 fragment. In the resistant population, the mutation C(3004) → G resulted in the substitution L(1002) → V (codon ctg → gtg) at the position equivalent to that of the housefly L925 in the domain II S5 helix. Additionally, the mutation F(1052) → L (codon ttc → ctc) at the position equivalent to that of the housefly F975 in the domain II P-loop connecting segments S5 and S6 was detected in both the resistant and sensitive populations. CONCLUSION: All individuals that survived the tau-fluvalinate treatment in the bioassay harboured the L(1002) → V mutation combined with the F(1052), while dead individuals from both the sensitive and resistant populations harboured mostly the L(1002) residue and either of the two residues at position 1052.