UBC Theses and Dissertations
Development and application of honey bee in vitro systems Chan, Man Yi Mandy
The honey bee, Apis mellifera, has become tightly linked to human agriculture as one of the most important pollinators. The recent honey bee population decline has raised global concerns of a pollination crisis, yet honey bee research lags far behind in available research tools compared to other model organisms, limiting the pace we can hope to advance our knowledge of honey bee biology and improve bee health. Therefore, the major goal of this thesis was to establish new tools and to improve some of the existing tools for honey bee research and then to demonstrate that these tools can be combined with other genetic and proteomic techniques to help us address questions on honey bee biology. First of all, honey bee primary cell cultures from various tissues could be established and maintained for at least four months. Embryonic cultures could be cryopreserved and also transduced with lentivirus to express EGFP. Proteomic analysis revealed biological pathways related to glucose metabolism and oxidative stress were significantly altered in primary cells during two weeks of cultivation. In addition to cell culture, in vitro larval rearing was also established and the use of various artificial diets was compared for ability to sustain growth. Basic larval diet was by far the most efficient formulation and it was applied to study honey bees’ response to American foulbrood (AFB) infection. RNA interference (RNAi) was used to silence prophenoloxidase, a gene implicated in bees’ resistance to AFB and a multiple reaction monitoring mass (MRM) spectrometry assay was developed to assess degree of knockdown. Although dosage response was observed in in vitro rearing for AFB infection, significant gene silencing could not be achieved. Overall, we established several in vitro systems, including cell cultures and in vitro larval rearing, for honey bee research and these systems in combination with lentiviral transduction, RNA interference, proteome analysis, and MRM assay could form a thorough analysis platform for future studies to improve our knowledge of honey bee biology.
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