Entomology, Department of


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A THESIS Presented to the Faculty of The Gradate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Entomology, Under the Supervision of Professor Nicholas J. Miller. Lincoln, Nebraska: August 2014

Copyright 2014 Ashley Yates


RNA interference (RNAi) is a naturally occurring phenomenon in eukaryotes in which a double-stranded RNA (dsRNA) suppresses the expression of a target gene. RNAi has markedly changed the way in which functional genetics studies are performed, especially in non-model organisms. In insects, the efficacy of RNAi is influenced by several factors, including the species and the methods of dsRNAs delivery.

The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is a pest of the plant family Solanaceae. RNAi in the CPB is of interest for potential use in insect management and as a tool to study the interaction with host plants. The efficacy of different methods of dsRNA delivery for the functional analysis of genes in the CPB was evaluated. As a proof of concept, the Laccase2 gene, which is responsible for pigmentation and sclerotization in beetles, was targeted for knockdown. The potential for systemic RNAi was evaluated for several dsRNA delivery methods. Results indicated that the potential for systemic and persistent RNAi exists for each of the methods evaluated here. A slight phenotypic effect of Laccase2 knockdown was observed in larvae fed dsRNAs on artificial diet, and a stronger phenotypic effect was exhibited by larval microinjection of dsRNAs. Additionally, qRT-PCR indicated RNAi knockdown by microinjection of embryos.

To gain an understanding of how RNAi genes respond to dsRNA, gene expression in response to dsRNA challenge was assessed using Illumina high throughput sequencing. Core RNAi genes were not differentially expressed following dsRNA challenge, although a handful of genes involved in the RNAi pathway were found to be upregulated.

Establishing dsRNA delivery methods for RNAi is a useful tool for studying insect-plant interactions in this system. Additionally, this research provides a glimpse into the RNAi pathway of the CPB, which can contribute to our understanding of RNAi variability among different insect species, and identify genes that are likely to respond to selection pressure if RNAi is employed for insect management.

Advisor: Nicholas J. Miller