EFFECTS OF BLOOD FEEDING ON THE TRANSCRIPTOME OF THE MALPIGHIAN TUBULES IN THE ASIAN TIGER MOSQUITO AEDES ALBOPICTUS Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master in Science in the Graduate School of The Ohio State University By Carlos J. Esquivel Palma, B.S. Graduate Program in Entomology The Ohio State University 2015 Master’s Examination Committee: Dr. Peter M. Piermarini, Advisor Dr. David L. Denlinger Dr. Andrew P. Michel Copyright by Carlos J. Esquivel Palma 2015 Abstract Mosquitoes are one of the major threats to human health worldwide. They are vectors of protozoans, arboviruses, and filarial nematodes that cause diseases in humans and animals. The Asian tiger mosquito Aedes albopictus is a vector of medically important arboviruses such as dengue fever, chikungunya fever, yellow fever, eastern equine encephalitis, La Crosse encephalitis, and West Nile fever. Control of these diseases often involves control of the mosquito vectors with chemical insecticides. However, the use of a limited number of chemicals with similar modes of actions has led to resistance in several mosquito species. The development of new insecticides with novel modes of action is considered a promising strategy to overcome resistance. The Piermarini lab has recently shown that the renal (Malpighian) tubules of mosquitoes are promising physiological targets to disrupt for killing mosquitoes via a novel mode of action. The Malpighian tubules play a critical role in the acute processing of blood meals, by mediating the rapid excretion of water and ions derived from the ingested blood. However, the physiological roles of Malpighian tubules during the chronic processing of blood meals after the initial diuresis is complete (~1-2 h after feeding) are not known. Therefore, the goal of this thesis was to characterize the transcriptomic dynamics of Malpighian tubules at 3 h, 12 h, and 24 h after blood feeding using next generation sequencing (RNA-Seq). ii In Chapter 2, we generated single-end sequences of cDNA libraries derived from Malpighian tubules of non-blood fed and blood fed A. albopictus (adult females) and mapped them to the existing genome of Aedes aegypti. We found molecular evidence for a dramatic functional transition occurring in the tubules after blood feeding by focusing on transcripts that showed sustained up- or down- regulation; i.e., two or more consecutive periods of differential expression. In brief, our results suggest that the Malpighian tubules switch from a tissue dedicated to salt and water excretion to one more focused on the detoxification and excretion of metabolic wastes associated with blood meal processing (e.g., heme, ammonia). In Chapter 3, we generated paired-end sequences of cDNA libraries derived from Malpighian tubules of non-blood fed and blood fed A. albopictus and assembled the first de novo transcriptome for mosquito Malpighian tubules. The data provide insights into functional processes enriched in the Malpighian tubules of non-blood fed mosquitoes and confirm the dramatic molecular changes we found in Chapter 2. In addition, we conducted physiological and biochemical assays on mosquitoes and isolated Malpighian tubules, respectively, under non-blood fed and blood fed mosquitoes to confirm whether the transcriptomic changes manifested in functional changes. As predicted by the molecules changes, we demonstrated that blood fed mosquitoes have a lower capacity to excrete urine compared to non-blood fed mosquitoes, and that Malpighian tubules from blood fed mosquitoes have a higher GST activity and amount of uric acid compared to tubules from non-blood fed mosquitoes. iii This study provides new insights into the molecular physiology of Malpighian tubules and their roles in the chronic processing of blood meals. In particular, this work provides 1) the first demonstration that blood feeding affects the Malpighian tubules, 2) the first Malpighian tubule transcriptome in mosquitoes, and 3) identifies potential insecticide targets that could be used to disrupt the renal processing of blood meals in mosquitoes. iv Dedication This work is dedicated to my parents for their unconditional love and support, to my sisters Dalia and Alice, and brother Nery, to my lovely family that trusted in me, and prays for me every day, and to a very special person that came to my life just at the right moment, Rian L. v Acknowledgments I would like to thank: Dr. Luis Cañas and Nuris Acosta for the invaluable opportunity that they provided me, my advisor Dr. Peter M. Piermarini for his patience and unconditional support, my friends Travis and Liu to make every single day, the best day, to all the Zamorano/OARDC friends, and to the wonderful Department of Entomology vi Vita 2007 …………………………………Associate Degree in Agriculture Central National School of Agriculture Bárcena, V. Nueva, Guatemala. 2011 ………………………………...Bachelor of Science, Science and Production Pan-American College of Agriculture, Zamorano Francisco Morazán, Honduras. 2012 - 2013………………………….Visiting Scholar, Department of Entomology The Ohio State University 2013 - present ………………………Graduate Teaching & Research Associate Department of Entomology The Ohio State University vii Publications Esquivel, C. J., Cassone, B. J., & Piermarini, P. M. (2014). Transcriptomic evidence for a dramatic functional transition of the Malpighian tubules after a blood meal in the Asian tiger mosquito Aedes albopictus. PLoS Neglected Tropical Diseases, 8(6). doi:10.1371/journal.pntd.0002929 Fields of Study Major Field: Entomology viii Table of Contents Page Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita .................................................................................................................................... vii List of Tables ...................................................................................................................... x List of Figures ................................................................................................................... xii 1. Chapter 1: Literature review ........................................................................................ 1 1.1. The Asian tiger mosquito Aedes albopictus. ......................................................... 1 1.2. Insecticide resistance in mosquitoes. ..................................................................... 2 1.3. Novel physiological targets to mitigate insecticide resistance. ............................. 3 1.4. Morphology and physiology of mosquito Malpighian tubules. ............................ 4 1.5. Mosquito studies characterizing the transcriptomic effects of blood feeding. ...... 9 1.6. Rationale and goals of the current study. ............................................................ 11 1.7. References ........................................................................................................... 16 2. Chapter 2: Transcriptomic evidence of a functional transition in the Malpighian tubules of Aedes albopictus following a blood meal. ....................................................... 24 2.1. Abstract: .............................................................................................................. 24 2.2. Introduction ......................................................................................................... 25 2.3. Materials and methods ......................................................................................... 27 2.4. Results ................................................................................................................. 34 2.5. Discussion ........................................................................................................... 41 2.6. Conclusions and significance .............................................................................. 53 2.7. References ........................................................................................................... 70 3. Chapter 3: De novo transcriptome of the Malpighian tubules of Aedes albopictus... 80 3.1. Abstract ............................................................................................................... 80 3.2. Introduction ......................................................................................................... 80 3.3. Material and methods .......................................................................................... 82 3.4. Results. ................................................................................................................ 92 3.6. Discussion ......................................................................................................... 108 3.7. Conclusions and significance ............................................................................ 122 3.8. References ......................................................................................................... 153 Addendum: Supplemental information (Excel files of Tables S1, S2, S3, S4, S5, S6, and S7) can be assessed via OhioLINK. ..................................................................... 163 4. Chapter 4: Summary and future directions .............................................................