RNAi-Mediated Gene Silencing in the Exotic Redbay Ambrosia Beetle, \u3ci\u3eXyleborus glabratus\u3c/i\u3e, and Insect-Fungal Interactions within the Laurel Wilt Complex

Laurel wilt disease (LWD) is a lethal vascular disease impacting lauraceous hosts caused by Harringtonia lauricola, the fungal symbiont of the redbay ambrosia beetle (Xyleborus glabratus Eichoff) (RAB) (Coleoptera: Curculionidae). LWD has caused tree mortality throughout the southeastern United States and is continuing to spread into new regions. Current management methods have not been successful in preventing spread, warranting investigation into innovative techniques including RNA interference (RNAi). Elongation factor-1 alpha (ef1a) and actin (act) were established as stably expressed reference genes after exposing beetles to different photoperiod, temperature, and dsRNA exposure. After RAB oral ingestion of double- stranded RNAs (dsRNAs) of three target genes (hsp, shi, and iap), mortality and changes in gene expression were assessed. Following exposure, significant mortality occurred in all three genes. Quantitative PCR (qPCR) was used to investigate changes in gene expression at different timepoints with significant changes occurring in all three target genes. Additionally, working in sassafras forests at the leading edge of the LWD range expansion, insects were evaluated for internal and phoretic presence of H. lauricola. Results show internal presence of H. lauricola in one hidden snout weevil and two granulate ambrosia beetles, demonstrating that other insects can potentially vector LWD