Functional Analysis of Cytochrome P450 Genes and Enzymes in the Termite Reticulitermes flavipes
This research project was pursued to better understand the function of cytochrome P450 enzymes in Reticulitermes flavipes and the potential involvement they might have in wood toxin metabolism. Termites are pests in many parts of the world due to their diet consisting solely of wood, also known as lignocellulose. Due to their economic cost, the discovery of novel forms of termite control is always of interest. It has been well documented that cytochrome P450s are one of nature’s central tools for oxidizing foreign compounds, which is an important process in xenobiotic metabolism. Lignocellulose is a complex compound that when metabolized, releases potentially toxic materials in the termite. Here, I investigated the roles of P450 enzymes in relation to wood toxin metabolism and other processes. Main objectives included, (1) determine extent to which P450s help termites survive toxins found in their wood diets, (2) determine caste, tissue, and diet-specific expression of R. flavipes P450s, and (3) functionally validate the contributions of candidate P450 genes with wood feeding. Several scientific approaches were used to complete these objectives using both organismal and molecular techniques. By conducting whole-organism bioassays with live termites, it was determined that termites died when treated with P450 inhibitors and then fed a wood diet. Based on the bioassay results, it was of interest to examine various aspects that would offer insight into P450 expression and functionality based on caste, tissue, wood/paper diets, hormone/primer pheromone treatments, and treatment with known P450 inhibitors. It was determined that in some cases gene expression was differentially expressed by some treatments or conditions, while it was either constitutively higher or unaffected in others. Observations made in this component of the study also offered new insights into the physiological functions of the tested genes. One consistent observation was that the select P450s were most highly expressed in the termite carcass rather than gut tissue. As a last step, RNAi was used to functionally validate the contributions of three select P450 genes: CYP15A1, CYP15F1 and CYP6AM3. Results link CYP15A1, and to a lesser extent, CYP6AM3 to possible metabolism of wood toxins and further show these genes as potentially valid targets for RNAi-based termiticides. However, data are not conclusive with regard to gene function due to the fact that potential gene interactions may be occurring between the genes of interest and the risk of competition and off-target effects associated with RNAi. This research supports the hypothesis of P450 involvement in wood toxin metabolism, but does not confirm any one specific gene is solely involved. What this research does suggest is that this is a very complex system and that gene interaction and various types of regulation are undoubtedly involved and that the P450 system of termites has value in the area of termiticide research and development.
Scharf, Purdue University.
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