Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia
ABSTRACT: Pathogen detection microarrays analyzing honeybee samples taken from the same hives over the course of a year, oligos correspond to specific pathogens or pathogen families of viruses, bacteria, fungi, protists, and other parasites Samples were analyzed with the E-Predict analysis package Twenty hives samples biweekly, Timepoint refers to sample collection week after start, where T01 refers to April 15, 2009. Hive refers to the specific hive sampled.
Project description:Pathogen detection microarrays analyzing honeybee samples taken from the same hives over the course of a year, oligos correspond to specific pathogens or pathogen families of viruses, bacteria, fungi, protists, and other parasites Samples were analyzed with the E-Predict analysis package Overall design: Twenty hives samples biweekly, Timepoint refers to sample collection week after start, where T01 refers to April 15, 2009. Hive refers to the specific hive sampled.
Project description:Pathogen detection microarrays analyzing honeybee samples taken after parasitization with a predatory fly, oligos correspond to specific pathogens or pathogen families of viruses, bacteria, fungi, protists, and other parasites Samples were analyzed with the E-Predict analysis package. Honey bees parasitized with the phorid fly Apocephalus borealis were screened for viral and non-viral pathogens by microarray.
Project description:The honey bee is a key pollinator in many agricultural operations as well as a model organism for studying the genetics and evolution of social behaviour. The Apis mellifera genome has been sequenced and annotated twice over, enabling proteomics and functional genomics methods for probing relevant aspects of their biology. One troubling trend that emerged from proteomic analyses is that honey bee peptide samples consistently result in lower peptide identification rates compared to other organisms. This suggests that the genome annotation can be improved, or atypical biological processes are interfering with the mass spectrometry workflow. First, we tested whether high levels of polymorphisms could explain some of the missed identifications by searching spectra against the reference proteome (OGSv3.2) versus a customized proteome of a single honey bee, but our results indicate that this contribution was minor. Likewise, error-tolerant peptide searches lead us to eliminate unexpected post-translational modifications as a major factor in missed identifications. We then used a proteogenomic approach with ~1,500 raw files to search for missing genes, new exons, and revive discarded annotations and identified over 2,000 new coding regions. These results will contribute to a more comprehensive genome annotation and facilitate continued research on this important insect.
Project description:Flenniken - Honey bee gene expression microarray experimental design<br>To minimize variability between samples all arrayed bees were obtained from a single brood comb from a naturally mated queen, therefore all the bees were age-matched half-sisters. The bees selected for microarray analysis of virus (Sindbis-eGFP) co-injected with either virus-specific-dsRNA (vs-dsRNA) or non-specific dsRNA (ns-dsRNA) exhibited the reduced virus phenotype that was seen in the majority of the bees assayed. The five representative bees from each condition (v, v+vs-dsRNA, v+ns-dsRNA, dsRNA, and mock/injected with buffer) selected for microarray analysis were free of pre-existing conditions (assessed by APM analysis) (Runckel, Flenniken et al., 2011). To facilitate gene expression comparisons between multiple treatment groups we utilized a reference-design strategy in which each Cy5-labeled experimental sample was hybridized with a standardized Cy3-labeled reference sample. A complex RNA mixture representing hundreds of bees of various ages exposed to difference treatment groups, served as the reference RNA sample.
Project description:Here, we examined the transcriptional and epigenetic (DNA methylation) responses to viral infection in honey bee workers. One-day old worker honey bees were fed solutions containing Israeli Acute Paralysis Virus (IAPV), a virus which causes muscle paralysis and death and has previously been associated with colony loss. Uninfected control and infected, symptomatic bees were collected within 20-24 hours after infection. Worker fat bodies, the primary tissue involved in metabolism, detoxification and immune responses, were collected for analysis. We performed transcriptome- and bisulfite-sequencing of the worker fat bodies to identify genome-wide gene expression and DNA methylation patterns associated with viral infection. There were 753 differentially expressed genes (FDR<0.05) in infected versus control bees, including several genes involved in epigenetic and antiviral pathways. DNA methylation status of 156 genes (FDR<0.1) changed significantly as a result of the infection, including those involved in antiviral responses in humans. There was no significant overlap between the significantly differentially expressed and significantly differentially methylated genes, and indeed, the genomic characteristics of these sets of genes were quite distinct. Our results indicate that honey bees have two distinct molecular pathways, mediated by transcription and methylation, that modulate protein levels and/or function in response to viral infections. Examination of epigenomic and transcriptomic antiviral responses to Israeli Acute Paralysis Virus in honey bees
Project description:Honey bee colonies were maintained in an apiary at the University of British Columbia. During the summer of 2018, 40 queens were reared from a single colony and half were allowed to open mate, while the other half were kept as virgins in plastic queen cages. Two weeks after emergence, the virgin queens were given two, eight-minute carbon dioxide treatments on 2 sequential days, then re-introduced to their nucleus colonies. This process stimulates virgin queens to begin laying71, and we conducted these treatments in order to minimize the physiological differences between virgin and mated queens. Virgin and mated queens were retrieved from their nucleus colonies and half of each (10) were subjected to heat-shock (42 ͦC, 2 h), and then maintained at 30 ͦC for 2 d. The other half were held only at 30 ͦC for 2 d. Four to six weeks after mating, the queens were anesthetized with carbon dioxide, beheaded, then their spermathecae (including the tracheal net) were removed with fine forceps. Both ovaries were also removed and weighed. During the same summer, 200 drones from a different colony in the same apiary were collected and maintained in the laboratory overnight at ambient temperature with excess syrup (50% sucrose). The next day, semen was harvested with glass capillaries according to the methods described above. Because many drones were not sexually mature, 60 semen samples (out of the 200 drones) were collected. Capillaries were placed in petri dishes and half (30) were heat-shocked as described above, then kept at 25 ͦC for 2 d. The other half were only kept at 25 ͦC for 2 d. Ten samples from each experimental group were used for sperm viability assays as described above.
Project description:Expression profiling of brains of free-flying forager bees and hive-restricted bees that rushed toward the hive entrance when the screen was removed, apparently to attempt to forage.
Project description:We propose a technology B-HIVE, which allows us to map HIV integrations in a cell population and measure their individual transcription. The principle of B-HIVE is to tag individual viral genomes with a unique barcode of 20 nucleotides that allows us to track the viral transcripts produced by each provirus in the cell population. Overall design: We performed two independent infection on Jurkat T cells by barcoded HIV (2 biological replicates). Each sample preparation and data analysis was done in parallel in 2 replicates.
Project description:Full title: Probing the pan genome of a foodborne bacterial pathogen Listeria monocytogenes: Implications for its niche adaptation, pathogenesis, and evolution Listeria monocytogenes is a foodborne bacterial pathogen well known for adaptability to diverse environmental and host niches, and a high fatality rate among infected, immuno-compromised individuals. Three genetic lineages have been identified within this species. Strains of genetic lineages I and II account for more than ninety percent of foodborne disease outbreaks worldwide, whereas strains from genetic lineage III are rarely implicated in human infectious for unknown, yet intriguing, reasons. Here we have probed the genomic diversity of 26 L. monocytogenes strains using both whole-genome sequences and a novel 385,000 probe pan-genome microarray, fully tiling the genomes of 20 representative strains. Using these methods to identify genes highly conserved in lineages I and II but rare in lineage III, we have identified 86 genes and 8 small RNAs that play roles in bacterial stress resistance, pathogenicity, and niche, potentially explaining the predominance of L. monocytogenes lineages I and II in foodborne disease outbreaks. Extending gene content analysis to all lineages revealed a L. monocytogenes core genome of approximately 2,350 genes (80% of each individual genome) and a pan-genomic reservoir of >4,000 unique genes. Combined gene content data from both sequences and arrays was used to reconstruct an informative phylogeny for the L. monocytogenes species that confirms three distinct lineages and describes the relationship of 9 new lineage III genomes. Comparative analysis of 18 fully sequenced L. monocytogenes lineage I and II genomes shows a high level of genomic conservation and synteny, indicative of a closed pan-genome, with moderate domain shuffling and sequence drift associated with bacteriophages is present in all lineages. In contrast with lineages I and II, notable genomic diversity and characteristics of an open pan-genome were observed in the lineage III genomes, including many strain-specific genes and a more complex conservation pattern. This indicates that the L. monocytogenes pan-genome has not yet been fully sampled by genome sequencing, and additional sequencing of lineage III genomes is necessary to survey the full diversity of this intriguing species and reveal its mechanisms for adaptability and virulence. This is a Listeria monocytogenes pan-genome tilling array designed using PanArray algorithm. 9 experimental strains (F2-569, M1-002, F2-208, J2-071, J1-208, W1-111, W1-110, F2-524, F2-501) vs reference (EGD-e) strain.
Project description:Finding the differences in gene expression in three regions of the brain, basal ganglia, white matter, and frontal cortex, in normal, HIV infected, HIV infected with neurocognitive impairment, and HIV infected with both neurocognitive impairment and encephalitis patients. Overall design: Twenty-four human subjects in four groups were examined A) Uninfected controls; B) HIV-1 infected subjects with no substantial neurocognitive impairment (NCI); C) Infected with substantial NCI without HIV encephalitis (HIVE); D) Infected with substantial NCI and HIVE. RNA from neocortex, white matter, and neostriatum was processed with the Affymetrix® array platform.