Regulatory and Evolutionary Mechanisms of Insecticide Resistance: Signaling Control, Target-Site Variation, and Management Across Major Pest Species

Abstract

This dissertation investigated mechanisms of insecticide resistance in two key pest species: the yellow fever mosquito Aedes aegypti and the poultry litter beetle Alphitobius diaperinus. Integrated toxicology, molecular biology, transcriptomics, genomic and genetic approaches, and field-based resistance surveys were used to identify regulatory pathways and uncover novel target-site variants associated with resistance. In addition, a piggyBac-based platform was developed that enables functional genetics research in M. domestica. I first synthesized current knowledge of metabolic and target-site resistance, highlighting limited understanding of upstream regulatory pathways in detoxification-based resistance and identifying genomic technologies capable of resolving these gaps. I then investigated resistance in Ae. aegypti using a pyrethroid-resistant Cas9 strain (PRCas9) generated by crossing a resistant Puerto Rico (PR) strain with susceptible Cas9 strain. Transcriptomic analyses on the PRCas9 strain revealed overexpression of cytochrome P450s, esterases, and the reactive oxygen species (ROS) -producing enzyme Nox4-Art. Functional assays showed that exogenous H₂O₂ increased resistance and expression of resistance-associated P450s, demonstrating that ROS signaling regulates detoxification and providing the first evidence that a signaling pathway modulates detoxification-based pyrethroid resistance in Ae. aegypti. This establishes a framework for dissecting upstream factors, molecular interactions, and regulatory networks that drive resistance evolution. To translate these mechanistic insights into control strategies, I evaluated an insecticide rotation approach in Ae. aegypti. Rotation of permethrin with malathion delayed but did not prevent resistance, as kdr mutations rapidly became fixed, highlighting the limitations of rotation alone and the need for genetically informed monitoring and diversified control strategies. Recognizing the broader relevance of multiple resistance mechanisms, I examined resistance in A. diaperinus across the Southeastern United States and identified widespread resistance to permethrin, imidacloprid, and malathion with variable susceptibility to other insecticides. Synergist assays and sequencing identified both metabolic and target-site mechanisms in A. diaperinun, providing molecular markers and guidance for integrated pest management. Finally, I developed a hyperactive piggyBac transposase enabling large DNA insertions in M. domestica, supporting future CRISPR-based studies. Overall, this research establishes a unified framework linking regulatory pathways, evolutionary dynamics, and control strategies to better understand and manage insecticide resistance across public health and agricultural systems.