Dataset Information

Transcriptomic Responses of the Honey Bee Brain to Infection with Deformed Wing Virus.

ABSTRACT: Managed colonies of European honey bees (Apis mellifera) are under threat from Varroa destructor mite infestation and infection with viruses vectored by mites. In particular, deformed wing virus (DWV) is a common viral pathogen infecting honey bees worldwide that has been shown to induce behavioral changes including precocious foraging and reduced associative learning. We investigated how DWV infection of bees affects the transcriptomic response of the brain. The transcriptomes of individual brains were analyzed using RNA-Seq after experimental infection of newly emerged adult bees with DWV. Two analytical methods were used to identify differentially expressed genes from the ~15,000 genes in the Apis mellifera genome. The 269 genes that had increased expression in DWV infected brains included genes involved in innate immunity such as antimicrobial peptides (AMPs), Ago2, and Dicer. Single bee brain NMR metabolomics methodology was developed for this work and indicates that proline is strongly elevated in DWV infected brains, consistent with the increased presence of the AMPs abaecin and apidaecin. The 1361 genes with reduced expression levels includes genes involved in cellular communication including G-protein coupled, tyrosine kinase, and ion-channel regulated signaling pathways. The number and function of the downregulated genes suggest that DWV has a major impact on neuron signaling that could explain DWV related behavioral changes.

SUBMITTER: Pizzorno MC

PROVIDER: S-EPMC7918736 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Transcriptomic Responses of the Honey Bee Brain to Infection with Deformed Wing Virus.

Pizzorno Marie C MC Field Kenneth K Kobokovich Amanda L AL Martin Phillip L PL Gupta Riju A RA Mammone Renata R Rovnyak David D Capaldi Elizabeth A EA

Viruses 20210212 2

Managed colonies of European honey bees (<i>Apis mellifera</i>) are under threat from <i>Varroa destructor</i> mite infestation and infection with viruses vectored by mites. In particular, deformed wing virus (DWV) is a common viral pathogen infecting honey bees worldwide that has been shown to induce behavioral changes including precocious foraging and reduced associative learning. We investigated how DWV infection of bees affects the transcriptomic response of the brain. The transcriptomes of ...[more]

PMID: 33673139

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Project description:Deformed wing virus (DWV) prevalence is high in honey bee (Apis mellifera) populations. The virus infects honey bees through vertical and horizontal transmission, leading to behavioural changes, wing deformity, and early mortality. To better understand the impacts of viral infection in the larval stage of honey bees, artificially reared honey bee larvae were infected with DWV (1.55 × 1010 copies/per larva). No significant mortality occurred in infected honey bee larvae, while the survival rates decreased significantly at the pupal stage. Examination of DWV replication revealed that viral replication began at 2 days post inoculation (d.p.i.), increased dramatically to 4 d.p.i., and then continuously increased in the pupal stage. To better understand the impact of DWV on the larval stage, DWV-infected and control groups were subjected to transcriptomic analysis at 4 d.p.i. Two hundred fifty-five differentially expressed genes (DEGs) (fold change ≥ 2 or ≤ -2) were identified. Of these DEGs, 168 genes were downregulated, and 87 genes were upregulated. Gene Ontology (GO) analysis showed that 141 DEGs (55.3%) were categorized into molecular functions, cellular components and biological processes. One hundred eleven genes (38 upregulated and 73 downregulated) were annotated by KO (KEGG Orthology) pathway mapping and involved metabolic pathways, biosynthesis of secondary metabolites and glycine, serine and threonine metabolism pathways. Validation of DEGs was performed, and the related gene expression levels showed a similar tendency to the DEG predictions at 4 d.p.i.; cell wall integrity and stress response component 1 (wsc1), cuticular protein and myo-inositol 2-dehydrogenase (iolG) were significantly upregulated, and small conductance calcium-activated potassium channel protein (SK) was significantly downregulated at 4 d.p.i. Related gene expression levels at different d.p.i. revealed that these DEGs were significantly regulated from the larval stage to the pupal stage, indicating the potential impacts of gene expression levels from the larval to the pupal stages. Taken together, DWV infection in the honey bee larval stage potentially influences the gene expression levels from larvae to pupae and reduces the survival rate of the pupal stage. This information emphasizes the consequences of DWV prevalence in honey bee larvae for apiculture.

Project description:Deformed wing virus (DWV) is a major bee pathogen that is actively transmitted by the parasitic mite Varroa destructor and plays a primary role in overwinter colony losses of Apis mellifera. Despite of the intense investigation driven by the unique environmental and economic importance of this essential pollinator species, the mechanisms underlying honeybee-DWV interactions are still poorly understood. Here we report on a group of honeybee proteins that were selectively immunoprecipitated by an antibody directed against DWV particles and present a functional analysis of a major component of this set of potential DWV interactors, which was identified as an arginine kinase (ArgK). ArgK RNA levels positively correlated with DWV load in field collected larvae and honeybees and significantly increased upon DWV injection in controlled laboratory conditions, indicating that the ArgK gene was upregulated by DWV infection. RNAi experiments performed in vitro to target ArgK gene expression resulted in reduced DWV viral load, thus demonstrating that ArgK function is required for DWV replication. Taken together, these data indicate that successful DWV infection relies on transcriptional regulation and active recruitment of host ArgK by this viral pathogen. Therefore, our findings open novel perspectives on the potential control of DWV pathogenicity by targeting its interaction with specific host proteins. Immunoprecipitated samples in quadruplicate were separated in a precast polyacrylamide gradient gel,run in MES buffer for 25 min at 200 V and the proteins were stained with the Imperial Coomassie stain. Each lane was divided into 6 bands, 48 bands in total, which where destained with 50mM ammonium bicarbonate and acetonitrile (ACN) 1:1 treated for cysteine reduction (10mM dithiothreitol at 56C for 45min) and alkylation (55mM iodoacetamide at RT for 30min in the dark), and for enzymatic digestion with 12.5ng ul trypsin at 37C overnight. The resulting peptide mixture was analyzed by LC MS MS using an Ultimate 3000 HPLC coupled with an Orbitrap Fusion Tribrid (Thermo Fisher Scientific, CA, United States). Peptides were desalted on a trap column and separated on a 19 cm long silica capillary, packed in house with a C18, 1.9um, 100 A resin. The analytical column was encased by a column oven (Sonation; 40C during data acquisition) and attached to a nanospray flex ion source. Peptides were separated on the analytical column by running a 95min gradient of buffer A (5 percent ACN, and 0.1 percent formic acid) and buffer B (95 percent acetonitrile and 0.1 percent formic acid), at a flow rate of 250nl min. The mass spectrometer operated in positive ion mode and full MS was acquired in the Orbitrap in the mass over charge 350 1550 scan range at 120K resolution. Data dependent acquisition was performed in top speed mode (3 s long maximum total cycle): the most intense precursors were selected through a monoisotopic precursor selection (MIPS) filter and with charge greater than 1, quadrupole isolated (1.6 mass over charge width) and fragmented by 30 percent higher energy collision dissociation. Product ion spectra were acquired in the ion trap with a rapid scan rate. Peptide spectra were analyzed with Proteome Discoverer 2.4 by the search engine Sequest HT using a mixed database containing Apis mellifera (reviewed and unreviewed 20.818 sequences; release 26 01 2022), homo sapiens (42.253 sequences, v2017 10 25) and Oryctolagus cuniculus (892 sequences, release 07 03 2022), and Deformed wing virus (1067 sequences, release 26 01 2022), all downloaded from Uniprot. Precursor and fragment mass tolerance were set to 15 ppm and 0.6 Da, respectively. Cysteine carbamydomethylation was set as static modification, while methionine oxidation and N-acetylation on protein terminus were set as variable modifications. Specific trypsin cleavages with maximum two miscleavages were admitted. Spectral matches were filtered at 1 percent false discovery rate (FDR), based on q values, and target decoy approach, using the Percolator node. Only master proteins were taken into account. Quantification was based on precursor intensity of unique and razor peptides. The Perseus software (1.6.15) was used to perform statistical analyses and evaluate differential protein expression. After contaminant removal (rabbit and human proteins), the abundance values were log2 transformed and filtered for containing at least four valid values in at least one group. Missing values were imputed with the constant value 13. A Students t-test was performed applying 250 randomizations, setting S0=2 and 0.01 FDR, and used to draw a Volcano plot to evaluate significantly different IP over IgG ratios.

Dataset Information

Transcriptomic Responses of the Honey Bee Brain to Infection with Deformed Wing Virus.

Publications

Transcriptomic Responses of the Honey Bee Brain to Infection with Deformed Wing Virus.

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