Undescribed viruses detected by RNA shotgun metagenomics sequencing of wild bees.

<p>Undescribed viruses detected by RNA shotgun metagenomics sequencing of wild bees.</p

Organization and characteristics of the segmented Ganda bee birus (GABV) genome.

<p>–<b>A)</b> similar workflow as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168456#pone.0168456.g002" target="_blank">Fig 2A</a>; predicted ORFs are indicated in yellow. Coverage of viral genomic RNA (green) is higher than complementary cRNA (red) for the L and S segments, but vice versa for the 3’ end of the M segment.–<b>B)</b> The GABV 5’ and 3’ terminal regions are depicted and show strong complementarity which is typical for segmented bunyaviruses. The terminal regions of two other phasmaviruses are identical in the first 10 residues (green/line) except for pos. 7 (red). The terminal repeats are present on each segment and are involved in the regulation of genome replication in bunyaviruses. The terminal regions of the M and S segments were not found.–<b>C)</b> similar as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168456#pone.0168456.g002" target="_blank">Fig 2C</a>; samples are RNA extractions from the same GABV-negative (-) or GABV-positive (+) female bee. All three segments are absent in a GABV-negative individual and therefore not part of the <i>O</i>. <i>cornuta</i> transcriptome. KIGV = Kigliuak phantom virus; WhMV-1 = Wuhan Mosquito Virus 1; RPL13a = Ribosomal Protein L13a.</p

Organization and characteristics of the unsegmented Scaldis River bee virus (SRBV) genome.

<p><b>-A)</b> High quality reads were mapped against the validated, near full-length genome sequence of SRBV using the CLC Genomic Workbench 8.5 mapping tool. Predicted ORFs that encode putative viral proteins were manually annotated (yellow). The readmap depicts the nucleotide coverage by viral genomic RNA (green) and complementary cRNA (red) reads. Note the relatively higher cRNA fraction towards the 3’ end of the genome.–<b>B)</b> Comparison of the genome organization of SRBV and its closest relative Shayang Fly Virus 1 (SyFV-1). RdRp (L), nucleoprotein (N), ORF2/VP2 (?) and glycoprotein (G) have similar positions and mass ranges.–<b>C)</b> RT-PCR amplification of 700–800 bp regions of the L, G, N genes and a housekeeping gene RPL13a in a SRBV-negative <i>Osmia cornuta</i> female (-) compared to a SRBV-positive female (+). Control samples C_L, C_G and C_N were viral RT-PCR products generated during sanger validation. The RPL13a control C_D is a DNA extraction sample of a bee leg. RPL13a intron-spanning primers were specifically designed to discriminate gDNA (313 bp) from cDNA (119 bp).</p

Differential expression of transporter encoding genes.

<p>Stacked percentage bar chart, showing the numbers of up- and down-regulated (putative) transporter encoding genes for T4-C4 (left) and T1-C1 (right), respectively, relative to the total numbers of (putative) transporter encoding genes within the <i>P. larvae</i> genome. The latter are indicated between square brackets. Round brackets: GO term numbers. GO terms were assigned with Blast2GO. White bars: down-regulation for T4-C4. Dark grey bars: up-regulation for T4-C4. Light grey bars: down-regulation for T1-C1. Black bars: up-regulation for T1-C1. Arrow heads: arbitrary GO term hierarchy (◂>⊲><>\raster(90%)="rg3"<>).</p

General overview of the microarray experiment, showing the total number of up- and down-regulated genes, for three selection criteria (columns) and four comparisons (rows).

<p>T1: test sample collected one hour after spiking with larval bodily fluid. T4: test sample collected four hours after spiking with larval bodily fluid. C1: control sample collected one hour after spiking with BHIT broth. C4: control sample collected four hours after spiking with BHIT broth. P: p-value. FC: fold change. Hyb: at least 75% of the different, hybridized probes for the same gene shows differential expression for a particular comparison.</p

Differential expression of metabolic genes.

<p>Stacked percentage bar chart, showing the numbers of up- and down-regulated (putative) metabolic genes for T4-C4 (left) and T1-C1 (right), respectively, relative to the total numbers of (putative) metabolic genes within the <i>P. larvae</i> genome. C: carbohydrate metabolism. The latter are indicated between square brackets. Round brackets: KO numbers (KEGG pathways). KEGG pathways were assigned with KAAS. White bars: down-regulation for T4-C4. Dark grey bars: up-regulation for T4-C4. Light grey bars: down-regulation for T1-C1. Black bars: up-regulation for T1-C1. E: energy metabolism. L: lipid metabolism. N: nucleotide metabolism. A: amino acid metabolism. ∼: KO:00061; KO:00071; KO:00592; KO:01040. *: KO:00360; KO:00350; KO:00380. °: KO:00410; KO:00430; KO:00450; KO:00460; KO:00480. ?: KO:00740; KO:00770; KO:00780; KO:00670; KO:00860; KO:00130. ‘:KO:00900; KO:00903; KO:00281; KO:00523; KO:01053; KO:01055; KO:00940; KO:00311; KO:00521. ‘’: KO:00362; KO:00627; KO:00625; KO:00622; KO:00633; KO:00642; KO:00643; KO:00930; KO:00363; KO:00621; KO:00626.</p

Effect of honey bee larval bodily fluid on <i>Paenibacillus larvae</i> bacterial density.

<p>OD590 (white bars) and CFU/ml (black bars) for T1, C1, T4 and C4. T1 (n = 12): test sample collected one hour after spiking with 5% larval fluids. T4 (n = 12): test sample collected four hours after spiking with 5% larval fluids. C1 (n = 11): control sample collected one hour after spiking with BHIT-broth. C4 (n = 12): control sample collected four hours after spiking with BHIT-broth. Between brackets (n): number of independent replicates. Error bars: standard deviations.</p

Effect of <i>Crithidia mellificae, Nosema ceranae and Varroa destructor</i> on honey bee colony winter losses.

<p>The presence of <i>C. mellificae</i>, <i>N. ceranae</i> and <i>V. destructor</i> in summer all increase later winter mortality (binomial probit model, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072443#pone-0072443-t002" target="_blank">Table 2b, p</a> = 0.01, 0.03 and 0.07, respectively). In addition, there is a synergistic effect of <i>C. mellificae</i> and <i>N. ceranae</i> on winter mortality (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072443#pone-0072443-t002" target="_blank">Table 2b, p</a> = 0.03). Cm = <i>C. mellificae</i>, Nc = <i>N. ceranae</i>.</p

GO-enrichment analysis for comparison T4–C4, showing the most specific over- and under-represented biological process GO-terms for both up- and down-regulation.

<p>NS, FE, P-value: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089175#pone-0089175-t002" target="_blank">Table 2</a>.</p