Methodology for Single Bee and Bee Brain 1H-NMR Metabolomics.

The feasibility of metabolomic 1H NMR spectroscopy is demonstrated for its potential to help unravel the complex factors that are impacting honeybee health and behavior. Targeted and non-targeted 1H NMR metabolic profiles of liquid and tissue samples of organisms could provide information on the pathology of infections and on environmentally induced stresses. This work reports on establishing extraction methods for NMR metabolic characterization of Apis mellifera, the European honeybee, describes the currently assignable aqueous metabolome, and gives examples of diverse samples (brain, head, body, whole bee) and biologically meaningful metabolic variation (drone, forager, day old, deformed wing virus). Both high-field (600 MHz) and low-field (80 MHz) methods are applicable, and 1H NMR can observe a useful subset of the metabolome of single bees using accessible NMR instrumentation (600 MHz, inverse room temperature probe) in order to avoid pooling several bees. Metabolite levels and changes can be measured by NMR in the bee brain, where dysregulation of metabolic processes has been implicated in colony collapse. For a targeted study, the ability to recover 10-hydroxy-2-decenoic acid in mandibular glands is shown, as well as markers of interest in the bee brain such as GABA (4-aminobutyrate), proline, and arginine. The findings here support the growing use of 1H NMR more broadly in bees, native pollinators, and insects.

Pollen DNA metabarcoding identifies regional provenance and high plant diversity in Australian honey.

Accurate identification of the botanical components of honey can be used to establish its geographical provenance, while also providing insights into honeybee (Apis mellifera L.) diet and foraging preferences. DNA metabarcoding has been demonstrated as a robust method to identify plant species from pollen and pollen-based products, including honey. We investigated the use of pollen metabarcoding to identify the floral sources and local foraging preferences of honeybees using 15 honey samples from six bioregions from eastern and western Australia. We used two plant metabarcoding markers, ITS2 and the trnL P6 loop. Both markers combined identified a total of 55 plant families, 67 genera, and 43 species. The trnL P6 loop marker provided significantly higher detection of taxa, detecting an average of 15.6 taxa per sample, compared to 4.6 with ITS2. Most honeys were dominated by Eucalyptus and other Myrtaceae species, with a few honeys dominated by Macadamia (Proteaceae) and Fabaceae. Metabarcoding detected the nominal primary source provided by beekeepers among the top five most abundant taxa for 85% of samples. We found that eastern and western honeys could be clearly differentiated by their floral composition, and clustered into bioregions with the trnL marker. Comparison with previous results obtained from melissopalynology shows that metabarcoding can detect similar numbers of plant families and genera, but provides significantly higher resolution at species level. Our results show that pollen DNA metabarcoding is a powerful and robust method for detecting honey provenance and examining the diet of honeybees. This is particularly relevant for hives foraging on the unique and diverse flora of the Australian continent, with the potential to be used as a novel monitoring tool for honeybee floral resources.

Demonstrating the potential of a novel spider venom-based biopesticide for target-specific control of the small hive beetle, a serious pest of the European honeybee.

The parasitic small hive beetle (Aethina tumida) feeds on pollen, honey and brood of the European honey bee (Apis mellifera); establishment in North America and Australia has resulted in severe economic damage to the apiculture industry. We report potential for the "in-hive" use of a novel biopesticide that is toxic to this invasive beetle pest but harmless to honeybees. Constructs encoding the spider venom neurotoxin ω-hexatoxin-Hv1a (Hv1a) linked to the N- or C-terminus of snowdrop lectin (GNA) were used to produce recombinant Hv1a/GNA and GNA/Hv1a fusion proteins. Both were similarly toxic to beetles by injection (respective LD50s 1.5 and 0.9 nmoles/g larvae), whereas no effects on adult honeybee survival were observed at injection doses of > 200 nmoles/g insect. When fed to A. tumida larvae, GNA/Hv1a was significantly more effective than Hv1a/GNA (LC50s of 0.52 and 1.14 mg/ml diet, respectively), whereas both proteins were similarly toxic to adults. Results suggested that the reduced efficacy of Hv1a/GNA against larvae was attributable to differences in the susceptibility of the fusion proteins to cleavage by gut serine proteases. In laboratory assays, A. tumida larval survival was significantly reduced when brood, inoculated with eggs, was treated with GNA/Hv1a.

Honeybees prefer novel insect-pollinated flower shapes over bird-pollinated flower shapes.

Plant-pollinator interactions have a fundamental influence on flower evolution. Flower color signals are frequently tuned to the visual capabilities of important pollinators such as either bees or birds, but far less is known about whether flower shape influences the choices of pollinators. We tested European honeybee Apis mellifera preferences using novel achromatic (gray-scale) images of 12 insect-pollinated and 12 bird-pollinated native Australian flowers in Germany; thus, avoiding influences of color, odor, or prior experience. Independent bees were tested with a number of parameterized images specifically designed to assess preferences for size, shape, brightness, or the number of flower-like shapes present in an image. We show that honeybees have a preference for visiting images of insect-pollinated flowers and such a preference is most-likely mediated by holistic information rather than by individual image parameters. Our results indicate angiosperms have evolved flower shapes which influence the choice behavior of important pollinators, and thus suggest spatial achromatic flower properties are an important part of visual signaling for plant-pollinator interactions.

SNPs selected by information content outperform randomly selected microsatellite loci for delineating genetic identification and introgression in the endangered dark European honeybee (Apis mellifera mellifera).

The honeybee (Apis mellifera) has been threatened by multiple factors including pests and pathogens, pesticides and loss of locally adapted gene complexes due to replacement and introgression. In western Europe, the genetic integrity of the native A. m. mellifera (M-lineage) is endangered due to trading and intensive queen breeding with commercial subspecies of eastern European ancestry (C-lineage). Effective conservation actions require reliable molecular tools to identify pure-bred A. m. mellifera colonies. Microsatellites have been preferred for identification of A. m. mellifera stocks across conservation centres. However, owing to high throughput, easy transferability between laboratories and low genotyping error, SNPs promise to become popular. Here, we compared the resolving power of a widely utilized microsatellite set to detect structure and introgression with that of different sets that combine a variable number of SNPs selected for their information content and genomic proximity to the microsatellite loci. Contrary to every SNP data set, microsatellites did not discriminate between the two lineages in the PCA space. Mean introgression proportions were identical across the two marker types, although at the individual level, microsatellites' performance was relatively poor at the upper range of Q-values, a result reflected by their lower precision. Our results suggest that SNPs are more accurate and powerful than microsatellites for identification of A. m. mellifera colonies, especially when they are selected by information content.

Typing of Melissococcus plutonius isolated from European and Japanese honeybees suggests spread of sequence types across borders and between different Apis species.

Melissococcus plutonius is an important pathogen of honeybee larvae and causes European foulbrood (EFB) not only in European honeybees (Apis mellifera) but also in other native honeybees. We recently confirmed the first EFB case in Japanese native honeybees (Apis cerana japonica) and isolated M. plutonius from this case. In this study, to obtain a better understanding of the ecology of M. plutonius and the epidemiology of EFB, we analyzed M. plutonius isolates that originated from European and Japanese honeybees in Japan using an existing multilocus sequence typing scheme. These analyzed Japanese isolates were resolved into six sequence types (STs), three of which were novel STs. Among these six STs, ST3 and ST12 were the two most common and found in isolates from both European and Japanese honeybees (or their environment). Moreover, these two STs were identified not only in Japan but also in other countries, suggesting the spread of some STs across borders and different honeybee species.