AN RNAI-BASED APPROACH TO IPS CALLIGRAPHUS MANAGEMENT: A PROMISING STRATEGY FOR FOREST PROTECTION

Author

Mary Wallace, University of KentuckyFollow

Author ORCID Identifier

https://orcid.org/0009-0002-7641-3023

Date Available

5-4-2026

Year of Publication

2024

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

As climate change leads to more frequent and severe extreme weather, host tree stress is increasing the occurrence of large-scale bark beetle outbreaks. One such beetle, Ips calligraphus (Germar), a native bark beetle in the southeastern United States, has traditionally been considered a secondary pest of pine trees. Management has relied on silvicultural practices that enhance forest resilience, enabling host trees to effectively use natural defenses to deter beetle infestation. However, during periods of forest stress caused by storms, droughts, human disturbance, and other factors, beetle outbreaks can cause considerable ecological and economic damage.

Addressing worsening I. calligraphus outbreaks will require innovative management approaches to augment traditional silvicultural practices. One promising molecular approach for pest management involves using exogenous double-stranded RNA (dsRNA) to manipulate the RNA interference (RNAi) pathway, an immune response and gene regulatory mechanism found in most eukaryotes. Exogenous dsRNA is processed by insect cellular machinery, resulting in degradation of targeted mRNA, and impairing target gene function. My research addresses essential foundational steps to bring RNAi technology to fruition, including proof of concept for gene silencing via ingested dsRNA, evaluation of chemosensory target genes, and investigation of a yeast associate for microbial delivery.

In Chapter 2, I assessed candidate reference gene stability in I. calligraphus under experimentally relevant conditions, a crucial step before demonstrating gene silencing using RT-qPCR. I used four stably expressed genes (16s rRNA, 28s rRNA, ef1a, and cad) identified in Chapter 2 to normalize gene expression in Chapters 3 and 4. In Chapter 3, I assembled an adult beetle transcriptome, identified RNAi pathway genes, and potential target genes based on successful silencing and mortality in other scolytines. I then administered dsRNA targeting the iap, shi, and hsp genes, and found dsIAP and dsSHI caused significant beetle mortality. Gene expression analysis revealed significantly lower iap mRNA levels in dsIAP treated beetles, but increased shi expression in dsSHI treated beetles. This indicates that inducing gene expression changes and mortality using ingested dsRNA could be a viable approach for I. calligraphus and warrants further refinement as a possible management tool.

Chapters 4 and 5 explore alternative approaches for target gene selection and dsRNA delivery. In Chapter 4, I annotated chemosensory genes using the transcriptome data generated in Chapter 3 and assessed sex-based differential expression of these genes. Then, I designed and administered dsRNA targeting the orco gene, a co-receptor critical to odorant sensing, to adult beetles at three different concentrations. Female beetles exhibited gene silencing at each of the three dsRNA concentrations, while male beetles showed variable responses from gene silencing, to increased gene expression. Female beetles treated with dsRNA were then assessed for responsiveness to male frass in an olfactometer

assay to evaluate behavioral changes. Despite exhibiting gene silencing at the concentration administered, the female beetles showed no change in behavioral response.

Chapter 5 investigates a novel potential dsRNA deployment method by transforming Ogataea pini, a yeast associated with I. calligraphus and other bark beetles, to express insecticidal iap dsRNA, completing the circle for evaluation of gene silencing in I. calligraphus from initial proof-of-concept to delivery for broadscale tree protection. In summary, my research contributes to the growing body of literature on RNAi technology in forest pest management and is the first to artificially activate the RNAi pathway in an Ips species. Additionally, the insights gathered on alternative target genes and delivery methods provide valuable information for refining this technology as RNAi-based tools progress toward practical field use.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2024.206

Funding Information

USDA APHIS AP20PPQS&T00C061

University of Kentucky

Kentucky Agricultural Experiment Station under McIntire-Stennis 2351197000

Recommended Citation

Wallace, Mary, "AN RNAI-BASED APPROACH TO IPS CALLIGRAPHUS MANAGEMENT: A PROMISING STRATEGY FOR FOREST PROTECTION" (2024). Theses and Dissertations--Entomology. 85.
https://uknowledge.uky.edu/entomology_etds/85