Investigating RNA interference as a biopesticide for Dendroctonus ponderosae

In western North America, the mountain pine beetle (Dendroctonus ponderosae, MPB) is the most destructive pest of pine trees, consequently causing ecological, economic, and socioecological impacts. Previous and current management techniques are ecologically and economically costly, creating a need for a low-cost, ecologically safe method for MPB population management. A molecular approach, RNA interference (RNAi), is being increasingly applied to pest management strategies due to target specificity and low production costs. The RNAi pathway is a naturally occurring anti-viral pathway that degrades invading genetic material. When co-opted, the RNAi pathway can be used to silence targeted genes in insects by disrupting cellular function. Developing RNAi to use in ecologically safe trap trees can create a novel tool for managing MPB populations. Existing and new gene targets were tested using microinjection (injection of dsRNA) and oral delivery methods for causing species-specific MPB mortality. Oral delivery involved the use of both in vitro synthesized dsRNA and heatinactivated, hpRNA-expressing yeast delivered through direct feeding and association with pine phloem. Success of RNAi initiation was determined using mortality observations alongside gene expression analysis. This study has identified promising gene targets and laboratory-based dsRNA delivery methods for RNAi initiation in emerged adult MPB. This study has also provided new insights into previously established dsRNA delivery methods. High mortality in all injected MPB suggests that dsRNA microinjection is too invasive for emerged adult MPB, despite being a successful delivery method in congeneric species. A previously successful method, submerging MPB in dsRNA for twelve hours, elicited an RNAi response in four of the six gene targets. However, the results were not replicated and showed variation between bioassays. To advance the laboratory-based delivery method and provide support for RNAi use in lethal trap trees, a phloem-based dsRNA delivery method was also tested. Successful ingestion, high MPB survival in controls, and some RNAi-induced mortality supports the use of phloem-based methods for future RNAi assays in MPB. Lastly, an investigative transcriptome analysis confirmed the presence of critical RNAi components and provided insights into the MPB response to the different delivery methods. The submerged MPB mainly responded metabolically by breaking down energy stores, and the phloem-fed MPB showed the opposite metabolic response by creating energy stores. This analysis further supports the phloem-based dsRNA delivery for future RNAi studies on MPB. Future RNAi studies in MPB should consider emergence timing and its impact on MPB health to reduce the confounding factors affecting MPB mortality.