Figure 1.
Honey bees are productively infected with a model virus, SINV-GFP.
(A) Honey bees were infected with SINV-GFP and fluorescence microscopy revealed increased amounts of GFP each day post-injection (d.p.i.) (GFP pixel counts quantified using ImageJ in lower right corner of each image) and represented graphically in (B). (C) SINV-GFP was detected in honey bee protein lysates from the injection site, thorax (Th), and distal sites (D; head and abdomen) each d.p.i. by Western blot analysis using α-GFP (green lower arrow) and a-actin (top arrow) antibodies. Western blot bands were quantified using ImageJ and the GFP:actin ratio was normalized to 1* at day 2 p.i..
Figure 2.
dsRNA-mediated reduction of virus in honey bees.
(A) Schematic of experiment. Honey bees were inoculated with SINV-GFP or co-injected with (B) nucleic acid treatments including: virus-specific dsRNA (SINV dsRNA); non-specific dsRNA corresponding to Drosophila C virus (DCV) sequence (DCV dsRNA); poly(I:C); NTPs; non-specific dsRNA corresponding to luciferase sequence (LUC dsRNA); or SINV dsDNA visualized on a 1% agarose gel. (C) Fluorescence microscope images of a mock-infected bee (left panel), a SINV-GFP infected bee (middle panel), or a bee infected with SINV-GFP in the presence of non-specific dsRNA DCV. (D) Western blot analysis using α-GFP and α-actin antibodies demonstrated reduced SINV-GFP in bee protein lysates pooled from ten bees treated with dsRNA (lanes 1, 2, and 5) and poly(I:C) (lane 3), but not in bees treated with NTPs (lane 4) or dsDNA (lane 6) compared to bees inoculated with virus alone (virus). Mock-infected (mock) bees injected with buffer had no detectable GFP. (E) Boxplot of GFP fluorescence (510 nm, relative arbitrary units) in individual honey bee lysates (10 bees per condition). A Welch two-sample t-test was performed to compare honey bee lysates from each treatment group to virus-infected bees and determined statistically significant differences between bees co-inoculated with virus-specific dsRNA SINV p=0.023, with non-specific dsRNA DCV p=0.020, and with poly(I:C) p=0.0038, whereas co-injection of NTPs p=0.41 or dsDNA p=0.36 were not significantly different from virus alone (virus). Bees injected with virus only (virus) exhibited greater fluorescence compared to mock-infected controls (m); p=0.00001. (F) Relative abundance of SINV-GFP in individual bees (5 per condition) from different treatment groups was assessed by RT-qPCR. The bees treated with virus only had the highest SINV copy number 3 days p.i. (1.65x106 copies + 4,100 copies per 500 ng RNA, ~1.65x108 per bee), as compared to those in which either virus-specific SINV dsRNA (1.66x105 +1,050) or non-specific DCV dsRNA (2.32x105 + 1,070) was co-injected with virus.
Figure 3.
Differentially expressed honey bee genes.
(A) Table of differentially expressed genes (DEGs) in virus-infected or dsRNA-treated bees as compared to mock-infected bees (adj. p-value < 0.05); the number of genes with increased (up) and decreased (down) expression are listed below each condition.
(B) Venn diagram illustrating the degree of similarity and uniqueness of DEGs (adj. p-value < 0.5) between virus-infected (virus) and dsRNA-treated bees (dsRNA).
Figure 4.
Gene Ontology analysis demonstrated reduced expression of immune genes and perturbation of cell-signaling, trafficking, and metabolism.
Differentially expressed genes (DEGs, adj. p-value < 0.05) in (A) virus-infected or (B) dsRNA-treated honey bees as compared to mock-infected bees were assigned gene ontology terms for biological process, molecular function, and cellular compartment (see Tables S3-4). Biological process assignments were further categorized into seven functional groups: immune response (red); transcription, splicing, rRNA processing and RNAi (blue); signaling (purple); trafficking (green); metabolism (yellow); translation and protein folding (orange); and chromatin regulation (teal). A small fraction of the DEGs (10-15%) belonged to other biological process gene ontologies (black) and a large fraction was not assigned (gray). Each pie chart represents 100% of the DEGs and gene numbers are reported in each section. *Each oligonucleotide pair was analyzed as a single gene and since multiple microarray probes corresponded to apidaecin variants the majority of annotated immune effectors in this Figure (7 of 10 in virus-infected bees and 8 of 12 in dsRNA) correspond with apidaecin 1.
Figure 5.
Quantitative PCR validation of a subset of DEGs.
Quantitative PCR (qPCR) was used to validate honey bee gene expression microarray results. (A) apidaecin 1 expression in virus-infected bees was 20% + 6 of mock-infected controls (112% + 30%) and dsRNA-treatment also reduced expression to 15% + 3% of mock-infected controls; t-test p=0.01 and p=0.01 respectively. (B) unc-80 expression in virus-infected bees (126% + 9%) was higher than mock-infected controls (101% + 4%) (p=0.046). (C) lethal (3) expression increased in response to dsRNA-treatment to 156% + 14 relative to its expression in mock-infected bees 101% + 6% (p=0.03. Standard SYBR qPCR was performed for apidaecin 1 gene expression, whereas Taqman probe and primer sets were employed for other assayed genes. qPCR reactions were performed as technical triplicates of five individual bee cDNAs (biological replicates) per condition; the expression of each gene-of-interest (GOI) was compared to the housekeeping gene (Rpl8) and then the expression level of each GOI in five individual bees per experimental condition was calculated as a percentage relative to its expression in individual mock-infected bees using the ΔΔC(t) formula (y-axis), error bars represent +/- standard error of the mean (SEM), and statistical significance was assessed using Student’s t-tests.