Author ORCID Identifier

https://orcid.org/0000-0002-5317-8350

Date Available

12-8-2022

Year of Publication

2022

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department/School/Program

Entomology

First Advisor

Dr. Lynne Rieske-Kinney

Abstract

The emerald ash borer (EAB), Agrilus planipennis (Coleoptera: Buprestidae), is a highly invasive phloem-feeding insect native to Asia. It has killed millions of ash trees (Fraxinus spp.) since its accidental introduction into North America, causing profound economic and ecological impacts. RNA interference (RNAi) or double-stranded RNA (dsRNA)-mediated gene silencing is an emerging biotechnology that is developing as an innovative tool for pest management. Exogenous dsRNA triggers the RNAi pathway, silences genes, and disrupts protein function, causing insect mortality. RNAi has proven effective in reducing target gene expression and causing mortality in EAB; however, two aspects stand as barriers to the deployment of RNAi as a viable pest management approach: its specificity and practical delivery.

I based my work in Chapter 2 on the Environmental Protection Agency risk assessment framework for genetically engineered crops. I designed feeding bioassays followed by gene expression studies to evaluate the spectrum of activity of dsRNAs targeting the EAB genes hsp, shi and sn-rnp in model insects representing five feeding guilds including herbivore, predator, detritivore, pollinator, and parasitoid; the last represented by the classical biological control agents currently deployed for EAB management in North America. Bioassays demonstrated no lethal effects on the model insects, suggesting a narrow spectrum of activity for the three EAB-specific dsRNAs evaluated. The gene expression analyses suggest potential sublethal effects on the model pollinator; however, I found no effects on insect survival.

In Chapter 3, I evaluated dsRNA delivery using fluorescently tagged dsRNA and confocal microscopy to provide a proof of concept of uptake, movement, and bioactivity of dsRNA in green, F. pennsylvanica, and tropical ash, F. uhdei, through root and/or petiole absorption. Chapter 4 describes my greenhouse study exposing 2-year old green ash seedlings to dsRNAs as a foliar spray; I demonstrate in planta spread and persistence of topically applied dsRNA up to 21 days with an accompanying reduction in EAB target gene expression and reduced EAB gallery areas, demonstrating insecticidal activity of dsRNA applied as a foliar spray. In Chapter 5, I describe a parallel study using a root soak application; uptake, translocation, and persistence is demonstrated for up to 30 days, but no bioassays were conducted. Overall, my data demonstrate the high specificity of dsRNAs designed to suppress EAB and provide proof of concept of topical application feasibility to deliver dsRNA to EAB through the host plant.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.397

Funding Information

This work was supported through funds provided by USDA APHIS AP20PPQS&T00C0032, the University of Kentucky, and the Kentucky Agricultural Experiment Station under McIntire-Stennis 2351197000.

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