Investigating the Viral Ecology of Global Bee Communities with High-Throughput Metagenomics

Bee viral ecology is a fascinating emerging area of research: viruses exert a range of effects on their hosts, exacerbate the impacts of other environmental stressors, and, importantly, are readily shared across multiple bee species in a community. However, our understanding of bee viral communities is limited, as it is primarily derived from studies of North American and European Apis mellifera populations. Here, we examined viruses in populations of A. mellifera and 11 other bee species from 9 countries, across 5 continents and Oceania. We developed a novel pipeline to rapidly, inexpensively, and robustly screen for bee viruses. This pipeline includes purification of encapsulated RNA/DNA viruses, sequence-independent amplification, high throughput sequencing, integrated assembly of contigs, and filtering to identify contigs specifically corresponding to viral sequences. We identified sequences corresponding to (+)ssRNA, (-)ssRNA, dsRNA, and ssDNA viruses. Overall, we found 127 contigs corresponding to novel viruses (i.e. previously not observed in bees), with 29 represented by \u3e0.1% of the reads in a given sample. These viruses and viral families were distributed across multiple regions and species. This study provides a robust pipeline for metagenomics analysis of viruses, and greatly expands our understanding of the diversity of viruses found in bee communities

Association of viral diversity with colony size and <i>Varroa</i>.

<p><b>A</b>. Colony size (the number of frames of bees) was not affected by viral diversity (the number of viruses in a colony), H(2) = 2.74, p = 0.254. <b>B</b>. However, colonies with different number of viruses had significantly different numbers of <i>Varroa</i> (H(2) = 13.10; p = 0.0014). Colonies with 1 or 2 viruses had significantly higher <i>Varroa</i> loads than colonies that had no viruses (p<0.05, Wilcoxon pairwise tests, different letters denote significant differences). The number of colonies in each group is indicated at the bottom of each bar. <i>Varroa</i> counts were converted to logarithmic scale.</p

Association of <i>Varroa</i> infestation with elevation and colony size.

<p><b>A</b>. Levels of <i>Varroa</i> mites were positively correlated with elevation, with colonies at higher elevations having significantly higher average numbers of <i>Varroa</i> (r(53) = 0.44, p = 0.001). <b>B</b>. Levels were also positively correlated with colony size ((48) = 0.35, p = 0.013). <i>Varroa</i> counts were converted to logarithmic scale.</p

Primers used for molecular analysis for identification of bee populations, pathogens and parasites.

<p>Abbreviations: Israeli acute paralysis virus (IAPV), acute bee paralysis virus (ABPV), black queen cell virus, (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), kashmir bee virus (KBV), and sacbrood virus (SBV). References: Arias MC and WS Sheppard WS (1996) <i>Molecular Phylogenetics and Evolution</i> 5: 557–566; Benjeddou et al. (2001) <i>Applied and Environmental Microbiol</i>ogy 67:2384–2387; Chen et al. (2005) <i>Applied and Environmental Microbiol</i>ogy 71(1):436–441; Di Prisco et. al. (2011) <i>Journal of General Virology</i> 92: 151–15; Klee et al. (2007). Journal of Invertabrate Pathology 96: 1–10. Ribiere et al. (2002) <i>Apidologie</i> 33: 339–351; Stoltz et al. (1995) <i>Journal of Apicultural Research</i> 34: 153–160.</p

Geographic location of surveyed apiaries.

<p>Twenty-four apiaries were surveyed throughout Kenya with an additional three apiaries (25–27), see supplemenatry material, surveyed for ecological effects on colony health. The location and numerical designation of the apiaries is indicated on the map.</p