ABSTRACT MITCHELL III, ROBERT DRAKE. Global Human Health Risks for Arthropod Repellents or Insecticides and Alternative Control Strategies. (Under the direction of Dr. R. Michael Roe). Protein-coding genes and environmental chemicals. New paradigms for human health risk assessment of environmental chemicals emphasize the use of molecular methods and human-derived cell lines. In this study, we examined the effects of the insect repellent DEET (N, N-diethyl-m-toluamide) and the phenylpyrazole insecticide fipronil (fluocyanobenpyrazole) on transcript levels in primary human hepatocytes. These chemicals were tested individually and as a mixture. RNA-Seq showed that 100 µM DEET significantly increased transcript levels for 108 genes and lowered transcript levels for 64 genes and fipronil at 10 µM increased the levels of 2,246 transcripts and decreased the levels for 1,428 transcripts. Fipronil was 21-times more effective than DEET in eliciting changes, even though the treatment concentration was 10-fold lower for fipronil versus DEET. The mixture of DEET and fipronil produced a more than additive effect (levels increased for 3,017 transcripts and decreased for 2,087 transcripts). The transcripts affected in our treatments influenced various biological pathways and processes important to normal cellular functions. Long non-protein coding RNAs and environmental chemicals. While the synthesis and use of new chemical compounds is at an all-time high, the study of their potential impact on human health is quickly falling behind. We chose to examine the effects of two common environmental chemicals, the insect repellent DEET and the insecticide fipronil, on transcript levels of long non-protein coding RNAs (lncRNAs) in primary human hepatocytes. While lncRNAs are believed to play a critical role in numerous important biological processes many still remain uncharacterized and their functions and modes of action remain largely unclear. RNA-Seq showed that 100 µM DEET significantly increased transcript levels for 2 lncRNAs and lowered transcript levels for 18 lncRNAs while fipronil at 10 µM increased transcript levels for 76 lncRNAs and decreased levels for 193 lncRNAs. A mixture of 100 µM DEET and 10 µM fipronil increased transcript levels for 75 lncRNAs and lowered transcript levels for 258 lncRNAs. Differentially expressed lncRNA genes were mapped to chromosomes, analyzed by proximity to neighboring protein-coding genes, and functionally characterized via gene ontology and molecular mapping algorithms. While further testing is required to assess the organismal impact of changes in transcript levels, initial analysis links several of the dysregulated lncRNAs to processes and pathways critical to proper cellular function. Tick Haller’s organ detects infrared light. The Haller’s organ (HO), unique to ticks and mites, is found only on the first tarsus of the front pair of legs. The current thinking is that the HO’s main function is chemosensation analogous to the insect antennae, but the functionality of its atypical structure (exclusive to the Acari) is unexplained. We provide the first evidence that the HO allows the American dog tick, Dermacentor variabilis, to respond to infrared (IR) light. Unfed D. variabilis adults with their HOs present were positively phototactic to IR. However, when the HOs were removed by amputation of the tarsi bearing the HOs, no IR response was detected. Ticks in these experiments were also attracted to white light with and without the HOs, but were only positively phototactic to white light when the ocelli (primitive eyes) were present. Covering the eyes did not prevent IR attraction. A TRPA1 receptor was characterized from a D. variabilis-specific HO transcriptome we constructed. This receptor was homologous to the transient receptor potential cation channel, subfamily A, member 1 (TRPA1) from the pit organ of the pit viper, python, and boa families of snakes, the only receptor identified so far for IR detection. The ability of ticks to use IR for host finding is consistent with its obligatory hematophagy and has practical applications in tick trapping and the development of new repellents. © Copyright 2017 Robert Drake Mitchell III All Rights Reserved Global Human Health Risks for Arthropod Repellents or Insecticides and Alternative Control Strategies by Robert Drake Mitchell III A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Entomology Raleigh, North Carolina 2017 APPROVED BY: _______________________________ _______________________________ Dr. R. Michael Roe Dr. Daniel Sonenshine Committee Chair _______________________________ _______________________________ Dr. Ernest Hodgson Dr. Marcé Lorenzen _______________________________ Dr. Dominic Reisig DEDICATION I dedicate this dissertation to my mother and father, Bob and Beulah Mitchell. Without your love and support I wouldn’t be the man I am today or had the opportunities you provided. Thank you from the bottom of my heart. Also, to my fiancé Tiffany and my sisters Marie and Chris for their love and support. ii BIOGRAPHY Robert Drake Mitchell III was born to Bob and Beulah Mitchell on January 8, 1980 during a blizzard in Norfolk, VA. He was the third child in the family and the only boy. He attended Norfolk Collegiate School in Norfolk, VA through the entirety of high school and continued his education at American University in Washington, DC. Soon after receiving a bachelor’s degree in Biology he attended Old Dominion University in Norfolk, VA in pursuit of a Master’s degree. Under the tutelage of Dr. Daniel E. Sonenshine he developed a love for his work as well as a respect for its importance. He attended Eastern Virginia Medical School for three years as a graduate student before moving to Raleigh, NC to pursue a Ph.D. in Entomology at North Carolina State University under the guidance of Dr. R. Michael Roe. iii ACKNOWLEDGEMENTS To my family and friends. I’d like to thank everyone that has supported me along the way, including my fiancé Tiffany Benzine, my mother Beulah Mitchell, my sisters Marie Davis and Christine Mitchell, and my father Bob Mitchell who passed away six years ago. I regret that he is not here to see me succeed, but I know that he always had faith in me and will cherish that feeling for the rest of my life. Also, my nieces and nephews, who I hope have success in their own lives. I’d also like to personally thank two mentors that I hope to emulate throughout my career, Dr. Daniel Sonenshine and Dr. R. Michael Roe. Without either of them I’d not be where I am today or have the dedication and appreciation for my work that I’ve developed over the years and learned from them. Their tireless efforts have provided so much for the scientific community and for myself as well. My committee members have also been instrumental in my growth and understanding so I’d like to thank them as well: Dr. Marcé Lorenzen, Dr. Ernest Hodgson, Dr. Dominic Reisig, Dr. R. Michael Roe, and Dr. Daniel Sonenshine. Each has helped me at critical steps along the way and also helped me to stay focused through good times and more difficult times. Finally, I’d like to thank those that I’ve worked with throughout the years and gotten to know very well. I have such an eclectic group of friend that have supported and challenged me throughout my education, including Anirudh Dhammi, Jiwei Zhu, Jaap van Kretschmar, Nick, Travanty, Ann Carr, Loganathan Ponnusamy, Grayson Cave, John Strider, Haley Thornton Sutton, Marcel Deguenon, Charles Apperson, Clyde Sorenson, Shane Ceraul, Sayed Khalil, Kevin Donohue, Xin Guo and so many others. iv TABLE OF CONTENTS List of Tables .......................................................................................................................... vii List of Figures .......................................................................................................................... ix Chapter 1: Impact of Environmental Chemicals on the Transcriptome of Primary Human Hepatocytes: Potential for Health Effects ..................................................................................1 Abstract ..........................................................................................................................2 Introduction ....................................................................................................................3 Materials and Methods ...................................................................................................5 Cell Culture and Treatments ..........................................................................................5 RNA Isolation, Quality Assessment, and Sequencing ...................................................7 Data Analysis .................................................................................................................8 Quantitative PCR (qPCR) Analysis .............................................................................10 Results ..........................................................................................................................12 DEET and Fipronil Exposure Alter Gene Expression in Primary Human Hepatocytes ..................................................................................................................12 DEET-Fipronil Mixture ...............................................................................................15 Impact of DEET, Fipronil and DEET plus Fipronil