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Item Honey bee host-virus interactions at the individual and cellular level(Montana State University - Bozeman, College of Agriculture, 2021) McMenamin, Alexander James; Chairperson, Graduate Committee: Michelle Flenniken; This is a manuscript style paper that includes co-authored chapters.Honey bees are important pollinators of the fruits, nuts and vegetable crops that feed our growing population. Unfortunately, honey bee colony losses have averaged 38% from 2008-2018. These losses are due to a variety of factors, including reduced quality forage, pesticide exposure in agricultural fields, parasites like the Varroa destructor mite, and pathogens. The most diverse group of pathogens effecting honey bees are small RNA viruses. Honey bees have evolved numerous strategies to restrict virus infection, including the RNA interference (RNAi) pathway. Bees infected with a model virus, Sindbis-GFP (SINV) have differential expression of hundreds of genes, including RNAi genes and several heat shock protein (HSP) encoding genes. Therefore, we hypothesized that heat shock proteins are antiviral in honey bees. To induce the heat shock response (HSR), SINV-infected bees were heat shocked at 42°C for 4 hours. Heat shock resulted in a 74-90% reduction in SINV RNA copies as compared to bees maintained at 32°C. Heat shocked and/or virus-infected bees had increased expression of several core HSR protein-encoding genes, but heat shock did not consistently result in the increased expression of RNAi genes (argonaute-2 and dcr-like). This indicates that heat shock proteins are contributing to an antiviral response. SINV-infected bees also had higher expression of a recently identified antiviral gene - bee antiviral protein-1 (bap1). Therefore, we further characterized bap1 using computation approaches including phylogenetic analysis, which determined that this gene is taxonomically restricted to Hymenoptera and Blatella germanica (the German cockroach). Structural predication programs indicated that bap1 is a highly disordered protein. Intriguingly, transcriptome and correlation analyses determined that bap1 was coexpressed with several genes implicated in antiviral immunity (i.e., ago2, tudor-sn and TEP7). Although the precise antiviral function of bap1 remains to be elucidated, we further developed experimental tools that will enable more incisive investigation of bap1 and other antiviral genes. Primary cultures of larval hemocytes (immune cells) and mixed-cell pupal tissue cultures supported productive replication of sacbrood virus, deformed wing virus, and Flock House virus. Infected pupal cell cultures exhibited virus-specific transcriptional responses in bap1, ago2, and dcr-like expression. Together, these data further elucidate honey bee antiviral immunity and provide new tools for studying honey bee host-virus interactions.Item Honey bee antiviral defense mechanisms at the individual and cellular level(Montana State University - Bozeman, College of Agriculture, 2021) Parekh, Fenali Mukesh; Chairperson, Graduate Committee: Michelle Flenniken; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Chemical stimulants and stressors impact the outcome of virus infection and immune gene expression in honey bees (Apis mellifera)' in the journal 'Frontiers in immunology' which is contained within this dissertation.; Alexander J. McMenamin was an author and Verana Lawrence and Michelle L. Flennikenwas were co-authors of the article, 'Investigating virus-host interactions in cultured primary honey bee cells' in the journal 'Insects' which is contained within this dissertation.; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Honey bee antiviral response to flock house virus infection' which is contained within this dissertation.; This dissertation contains an article of which Fenali Mukesh Parekh is not the main author.Honey bees are important pollinators of fruit, nut, and vegetable crops that constitute a large proportion of the human diet. Unfortunately, annual honey bee colony losses are high, averaging 38% from 2008-2018 in the United States. Honey bee colony losses are attributed to multiple factors, including pathogens and chemical exposure. Virus incidence and abundance have been associated with colony losses. The majority of honey bee viruses are positive-sense single stranded RNA viruses. Honey bees antiviral defense include RNA interference (RNAi), a double-stranded RNA (dsRNA) triggered sequence-specific post-transcriptional silencing mechanism and a non-sequence specific dsRNA-triggered pathway. In addition, signal transduction cascades include the Toll, Imd, and Jak/STAT pathways that promote the expression of honey bee immune response genes that are also induced in response to virus infections. To investigate the impact of chemical exposure on honey bee immune responses and virus infections, we infected bees with a panel of viruses including two model viruses (i.e., Flock House virus (FHV) and Sindbis-GFP) and a naturally infecting honey bee virus, deformed wing virus (DWV) and fed them sucrose syrup containing either thyme oil, a beekeeper applied fungicide Fumagilin-B ®, or the insecticide clothianidin. We determined that bees fed thyme oil augmented sucrose syrup exhibited greater expression of key immune genes, i.e., ago2, dcr-like, abaecin, hymenoptaecin, and vitellogenin and reduced virus abundance compared to virus-infected bees fed sucrose syrup. Whereas, virus-infected honey bees fed diets containing fumagillin or clothianidin exhibited reduced expression of key immune genes and higher virus abundance suggesting that chemical stressors act as immunosuppressors in honey bees. To understand the interplay of viruses and host cell gene expression more precisely, we cultured primary honey bee cells derived from larvae (i.e., hemocytes, immune cells) or pupae (i.e., mixed cell population including epithelial cells, adipocytes, muscle cells, hemocytes) and demonstrated that these cells supported replication of sacbrood virus, DWV, and FHV. Expression of select immune genes, including bap1, ago2, and dcr-like, in virus-infected honey bee cells was similar to expression in individual bees and varied for each virus. Together, these data further our understanding of the honey bee antiviral defense network and provide new tools for studying honey bee host-virus interactions.