Date of Award

5-2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Juan Jurat-Fuentes

Committee Members

Albreht von Arnim, Ranjan Ganguly, Debra Miller

Abstract

Homeostasis of the intestinal epithelium in Heliothis virescens is mediated by the proliferation and differentiation of multipotent intestinal stem cells (ISCs) that lie adjacent to the basal lamina. In response to extrinsic and intrinsic signals, ISC proliferation and differentiation promotes epithelial growth and regeneration following the loss of integrity. We tested the in vivo effects of the ISC mitogen, a [alpha]-arylphorin, on ISC proliferation and the morphological changes of the midgut during larval development. Additionally, we examined how these changes affected the intestinal epithelium response to Cry1Ac toxin from Bacillus thuringiensis. Histological and in vitro evidence supported two distinct mechanisms whereby ingestion of -arylphorin reduced Cry1Ac toxicity in H. virescens larvae.

To characterize the role of alkaline phosphatase as a Cry toxin receptor in the H. virescens larval midgut, we used mechanistically distinct double-stranded RNA (dsRNA) delivery strategies to activate RNA-interference (RNAi) and induce transient silencing of midgut alkaline phosphatase (HvmALP) prior to exposing larvae to Cry1Ac toxin. Quantitative and qualitative analyses of gene and protein expression supported that hemocoelic microinjections resulted in the largest reduction in HvmALP protein expression and, furthermore, that bypassing physiological barriers such as the intestinal epithelium may enhance the efficiency of target gene silencing.

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